(Bacterio)chlorophyll photosensitizers for treatment of eye diseases and disorders

ABSTRACT

An ophthalmic composition is provided, comprising chlorophyll or bacteriochlorophyll compounds for photodynamic treatment (PDT) of diseases, disorders and conditions associated with corneal or scleral anomalies, such as corneal thinning and scleral stretching.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a National Stage of International Application No.PCT/IL2012/050325, filed Aug. 23, 2012, which claims the benefit under35 U.S.C. §119(e) of U.S. Provisional Application No. 61/526,414, filedAug. 23, 2011, the contents of all of which are incorporated herein intheir entireties by reference thereto.

TECHNICAL FIELD

The present invention is in the fields of ophthalmology and photodynamictherapy (PDT) and relates to photodynamic therapy of diseases, disordersand conditions associated with corneal or scleral anomaly, usingphotosensitizers, particularly water-soluble chlorophyll andbacteriochlorophyll compounds.

BACKGROUND ART

Treatment with riboflavin (RF) followed by ultraviolet A (UVA, 370 nm)illumination results in cornea stiffening and sclera stiffeningpresumably because of collagen crosslinking (CXL). This has increasinglybeen used for halting the progression of keratoconus and post refractivelaser surgery corneal ectasia (Wollensak et al., 2003(a); Hafezi et al.,2007; Raiskup-Wolf et al., 2008). However, there are several drawbacksto this treatment: (1) The prolonged time of RF treatment (30 min); (2)The prolonged eye exposure to UVA irradiation (30 min), and finally (3)Toxicity to keratocytes (Wollensak et al., 2004(a); Wollensak et al.,2004(b); Wollensak, 2010(a)) and corneal endothelial cells (Wollensak etal., 2003(c); Spoerl et al. 2007), making treatment of corneas thinnerthan 400 microns problematic (Hafezi et al., 2009; Wollensak, 2010(b)).Hence, there is a need for a safer treatment that can stiffen the corneawith a lesser risk to the patient (Avila and Navia, 2010;WO/2008/052081). One possibility is to use photosensitizers that inflictcornea stiffening upon illumination at the near infra red (NIR) usingbacteriochlorophyll derivatives as photosensitizers.

Myopia, also termed nearsightedness is a refractive defect of the eye inwhich collimated light produces the image focus in front of the retinawhen accommodation is relaxed. The global prevalence of myopia has beenestimated from 800 million to 2.3 billion. In some countries, such asChina, India and Malaysia, up to 41% of the adult population is myopicto −1 dpt and about 80% to −0.5 dpt. Myopia has been related withstretching of the collagenous sclera. Elongation of the globe occurs inthe posterior segment of the globe and involves the sclera. Such globeelongation causes myopic progression in predisposed myopic children andadolescents. It usually slows down and stops during the third decade oflife, when maturation of body tissues occurs with natural stiffening.This stiffening is related to glycation mediated cross linking.

At present, there is no effective treatment to stop myopic progressionand reduce visual loss caused by degenerative myopia. Surgical solutionsto arrest myopic progression by applying reinforcement belts around theeye, and suturing them to the sclera, were reported. These surgicalsolutions were controversial and technically challenging, and did notgain popularity. The critical age of intervention is during childhood orearly adolescence. Thus, a simpler approach to stiffen the sclera shouldbe applied.

Since progression of myopia is associated with elongation of theposterior segment of the eye and subsequent stretching of the sclera andchorioretinal tissues, stiffening of the sclera by collagencrosslinking, is expected to retard/stop the progression of the diseaseand related disorders such as macular stretching and atrophy or bleedingand visual loss. Wollensak and Spoerl reported the use of RF/UVAtreatment to achieve such crosslinking and strengthening in human andporcine sclera in vitro. The crosslinking stiffening was demonstrated invivo on rabbits, and was shown to last several months. This treatmentcan be applied to arrest myopic progression.

However, such treatment is subjected to the UVA risks which might behazardous. In addition, the tissue penetration of UV radiation islimited. Illumination of the sclera with UV requires external approachand necessitates surgical exposure. There is therefore, a need foralternative photosensitizers that can induce collagen crosslinking witha safer and better penetrating wavelength at the red or near infrared(NIR).

A non hazardous light with deeper tissue penetration, like NIR inbacteriochlorophyll (BChl)-based PDT has been shown to provide efficientand safe anti-cancer treatments in oncology and age related maculardegeneration in the eye (AMD) (U.S. Pat. No. 7,947,672, WO 2005/120573).

Application of novel water soluble chlorophyll (Chl) andbacteriochlorophyll (BChl) derivatives as sensitizers in PDT has beenreported by the present inventors in recent years (U.S. Pat. No.7,947,672; WO 2005/120573; Ashur et al. 2009; Mazor et al. 2005; Brandiset al., 2005) and by others (Moore et al., 2009; Bourges et al., 2006;Berdugo et al. 2008). Upon NIR illumination these water solublederivatives generate O₂ ⁻ and .OH radicals (Ashur et al. 2009; Mazor etal. 2005; Brandis et al., 2005; Vakrat-Haglili et al., 2005) and havebeen used so far in vascular-targeted photodynamic therapy (VTP) ofcancers in preclinical (Mazor et al. 2005) and advanced clinical trialsof prostate cancer therapy (currently in Phase III) (Trachtenberg J etal. 2007; Lepor H. 2008; Moore et al., 2009), following i.v.administration to the treated patients. The effective generation ofoxygen radicals as precursors of protein crosslinking (Liu et al.,2004), and the clinical experience established with water soluble Bchlsand Chls derivatives, makes these sensitizers potential candidates forapplication in therapy that is mediated by collagen crosslinking,particularly corneal and scleral stiffening upon NIR illumination aftertopical application.

SUMMARY OF THE INVENTION

It has now been found in accordance with the present invention that thewater soluble (bacterio)chlorophyll derivatives described in U.S. Pat.No. 7,947,672 and WO 2005/120573 enhance, upon NIR illumination,stiffening of the cornea and sclera of rabbit eye after topicalapplication. Non-limiting exemplifying results are disclosed herein forex vivo and in vivo treatment of rabbit eyes with certain water solublesulfonated bacteriochlorophyll derivatives. Treatment of corneas andsclera of rabbit eyes with these photosensitizers appeared safe andincreased significantly the biomechanical strength of the cornea and thesclera.

The present invention thus relates to the use of chlorophyll andbacteriochlorophyll derivatives for minimally invasive photodynamictherapy (PDT) of diseases, disorders and conditions associated withcorneal or scleral anomaly, particularly with corneal thinning orscleral stretching.

In a main aspect, the present invention provides ophthalmic compositionsfor use in PDT of the eye comprising a (bacterio)chlorophyll derivativeof the formula I, II or III herein, that significantly enhance cornealand scleral stiffening upon NIR irradiation.

In another aspect, the present invention relates to a method fortreatment of eye diseases, disorders and conditions, particularlydisorders associated with thinning of the cornea such as keratoconus,raised intraocular pressure and corneal ectasia caused by trauma, anddisorders associated with eye globe elongation such as myopia andmacular stretching.

The present invention further provides a method for preventing a cornealand/or scleral disease or weakening before, during or afterinterventional procedures.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is an illustration of a corneal strip clamped at a distance of 6mm between the jaws of a microcomputer-controlled biomaterial tester.

FIG. 2 is an illustration of a central corneal disc, 8 mm in diameter,mounted on a glass slide that was placed obliquely in a quartz cuvettefor fluorescence spectroscopy measurements after WST11/NIR and RF/UVAtreatment.

FIG. 3 are optical absorption spectra (755 nm) of WST11 accumulated inde-epithelialized corneas of rabbit exposed to a solution of WST11 insaline for 10, 20 and 30 minutes.

FIGS. 4A-4H are fluorescence microscope pictures (FIGS. 4A, 4B, 4E, 4F)and graphs related thereto (FIGS. 4C, 4D, 4G, 4H), showing fluorescenceintensity versus penetration depth into de-epithelialized rabbit corneasthat were exposed ex-vivo to WST11 in saline for 10 minutes (4A and 4C)and 30 minutes (4B and 4D), and to a solution of WST11 and dextran-500for 10 minutes (4E and 4G), and 30 minutes (4F and 4H). Fluorescence ofcentral corneal sagittal slices (12 μm) was detected at 760 nm uponexcitation at 740 nm.

FIG. 5 are optical absorption spectra of WST11 from corneas ex-vivotreated with WST11 solution for 20 minutes prior to illumination at 755nm, 10 mW/cm², for the specified time durations.

FIG. 6 is an electron spin resonance (ESR) spectrum of α-(4-pyridylN-oxide)-N-tert-butylnitrone (4-POBN, 65 mM) in solution containingethanol (8%) and WST11 (black) or RF (light blue), following NIR or UVAirradiation, respectively. The black arrows shows the quartet formedupon trapping singlet oxygen by 4-POBN, and the stars shows thedouble-triplet formed upon trapping hydroxyl and superoxide radicals.

FIG. 7 are ESR spectra of 4-POBN obtained form ex-vivo WST11/NIR (black)or RF/UVA (light blue) treated corneas, following immersion in a 4-POBN(65 mM) ethanol-(8%) solution.

FIGS. 8A-8B are graphs presenting ex vivo stress-strain measurementsvalues of corneal stiffness in ultimate stress units (8A) and Young'smodulus (8B) of corneas after 30-min. incubation with WST11 followed by30-min. NIR illumination (n=10), or incubation with RF-D solutionfollowed by UVA irradiation (RF-D/UVA).

FIGS. 9A-9B are graphs presenting in vivo stress-strain measurementsvalues of corneal stiffness in ultimate stress units (9A) and Young'smodulus (9B), 1 month after treating rabbit corneas with WST11 (2.5mg/ml) for 10, 20, or 30 minutes, followed by 30-min NIR illumination(755 nm, 10 mW/cm²), or after RF/UVA treatment.

FIGS. 10A-10B are graphs presenting in vivo stress-strain measurementsvalues of corneal stiffness in ultimate stress units (10A) and Young'smodulus (10B), 1 month after treating rabbit corneas either with WST11(2.5 mg/ml) in saline without dextran (WST11), or WST11 with dextran 500(WST-D) for 20 minutes, followed by 30-min NIR illumination (755 nm, 10mW/cm²), or after treatment with RF with or without dextran, followed by30-min UVA irradiation. Control-untreated eyes.

FIGS. 11A-11C are histological sections of rabbit corneas stained withhematoxylin-eosin two days (11A-11C) or 1 week (11D) post in vivoWST11/NIR illumination treatment (×20 magnification). 11A: control; 11B:cornea treated with WST11 in saline (WST11-S/NIR protocol); 11C: corneatreated with WST11 and dextran (WST11-D/NIR); 11D: 1 week afterWST11-D/NIR treatment.

FIGS. 12A-12B are histological sections of rabbit corneas stained forapoptosis 1 day after in vivo treatment with WST11 in saline followed byNIR illumination. 12A—Control, untreated cornea; 12B WST11-S/NIR treatedcornea.

FIG. 13 presents fluorescence spectra of rabbit corneas treated withWST11 and NIR irradiation (WST11/NIR), or treated with riboflavin andUVA irradiation (RF/UVA).

FIG. 14 is a fluorescence microscope picture of rabbit sclera showingpenetration of WST11.

FIGS. 15A-15B are graphs presenting ex vivo stress-strain measurementsvalues of upper equatorial sclera stiffness in ultimate stress units(15A) and Young's modulus (15B) of corneas after 30-min incubation withWST11 followed by 30-min direct NIR illumination (755 nm, 10 mW/cm²)onto the posterior treated sclera. Control—untreated lower equatorialsclera.

FIGS. 16A-16B are graphs presenting ex vivo stress-strain measurementsvalues of upper equatorial sclera stiffness in ultimate stress units(15A) and Young's modulus (15B) of corneas after 20-min incubation withWST11 followed by 30-min NIR illumination (755 nm, 10 mW/cm²) throughthe anterior cornea. Control—untreated lower equatorial sclera.

FIGS. 17A-17B are schematic illustration of the apparatus used forex-vivo drug delivery to the sclera and external or direct illumination(17A) and illumination through the anterior cornea (17B).

FIGS. 18A-18C are illustrations of the three mirror fundus lensapparatus used for NIR illumination of the posterior treated sclera of arabbit eye by illuminating the anterior cornea.

DETAILED DESCRIPTION

Definitions and Abbreviations

Bchl a: bacteriochlorophyll a: pentacyclic 7,8,17,18-tetrahydroporphyrinwith a 5^(th) isocyclic ring, a central Mg atom, a phytyl orgeranylgeranyl group at position 17³, a COOCH₃ group at position 13², anH atom at position 13², methyl groups at positions 2, 7, 12, 18, anacetyl group at position 3, and an ethyl group at position 8, hereincompound 1; Bphe: bacteriopheophytin a (Bchl in which the central Mg isreplaced by two H atoms); Bpheid: bacteriopheophorbide a (the C-17²-freecarboxylic acid derived from Bphe without the central metal atom); Chl:chlorophyll; Rhodobacteriochiorin: tetracyclic7,8,17,18-tetrahydroporphyrin having a -CH₂CH₂COOH group at position 17,a -COOH at position 13, methyl groups at positions 2, 7, 12, 18, ethylgroup at position 8 and vinyl as position 3; Pd-Bpheid:Pd-bacteriopheophorbide a; WST 11: palladium 3¹-oxo-15-methoxycarbonylmethyl-rhodobacteriochlonn 13¹-(2-sulfoethyl) amide dipotassium salt;ROS: reactive oxygen species; NIR: near infrared; RF: riboflavin.

IUPAC numbering of the bacteriochlorophyll derivatives is usedthroughout the specification.

MODES FOR CARRYING OUT THE INVENTION

It has been found by the present inventors that certain water solublechlorophyll and bacteriochlorophyll derivatives penetrate sclera andde-epithelialized cornea fairly fast and in a time dependent manner, andupon sensitization by the appropriate irradiation induce consistent andsignificant stiffening of the cornea and the sclera both ex vivo and invivo. The fact that these photosensitizers were photochemically activewas reflected in their continuous bleaching and spectra modificationinto their oxidized form during illumination.

Thus, it is a main object of the present invention to providepharmaceutical compositions comprising a chlorophyll orbacteriochlorophyll photosensitizer for use in minimally invasivephotodynamic therapy (PDT) of diseases, disorders and conditionsassociated with corneal or scleral anomaly. Particularly, thepharmaceutical composition provided by the invention is for treatment ofcorneal thinning and/or sclera stretching.

In preferred embodiments, the photosensitizer useful for the purpose ofthe invention is a water soluble (bacterio)chlorophyll of the formula I,II or III:

wherein

M represents 2H or an atom selected from the group consisting of Mg, Pd,Pt, Zn, In, Gd and Yb;

X is O or N—R₇;

R₁, R′₂ and R₆ each independently is Y—R₈, —NR₉R′₉ or —N⁺R₉R′₉R″₉A⁻;

Y is O or S;

R₂ is H, OH or COOR₉;

R₃ is H, OH, C₁-C₁₂ alkyl or C₁-C₁₂ alkoxy;

R₄ is —CH═CR₉R′₉, —CH═CR₉Hal, —CH═CH—CH₂—NR₉R′₉, —CH═CH—CH₂—N⁺R₉R′₉R″₉A⁻, —CHO, —CH═NR₉, —CH═N⁺R₉R′₉A⁻, —CH₂—OR₉, —CH₂—SR₉,—CH₂—Hal, —CH₂—R₉, —CH₂—NR₉R′₉, —CH₂—N⁺R₉R′₉R″₉A⁻, —CH₂—CH₂R₉,—CH₂—CH₂Hal, —CH₂—CH₂OR₉, —CH₂—CH₂SR₉, —CH₂—CH₂—NR₉R′₉,—CH₂—CH₂—N⁺R₉R′₉R″₉A⁻, —COCH₃, C(CH₃)═CR₉R′₉, —C(CH₃)═CR₉Hal,—C(CH₃)═NR₉, —CH(CH₃)═N⁺R₉R′₉A⁻ ⁻ , —CH(CH₃)—Hal, —CH(CH₃)—OR₉,—CH(CH₃)—SR₉, —CH(CH₃)—NR₉R′₉, —CH(CH₃)—N⁺R₉R′₉R″₉A⁻, or —C≡CR₉;

R′₄ is methyl or formyl;

R₅ is O, S, N—R₉, N⁺R₉R′₉A⁻, CR₉R′₉, or CR₉—Hal;

R₇, R₈, R₉, R′₉ and R″₉ each independently is:

(a) H;

(b) C₁-C₂₅ hydrocarbyl;

(c) C₁-C₂₅ hydrocarbyl substituted by one or more functional groupsselected from the group consisting of halogen, nitro, oxo, OR, SR,epoxy, epithio, —CONRR′, —COR, COOR″, —OSO₃R, —SO₃R″, —SO₂R, —NHSO₂R,—SO₂NRR′, —NRR′, ═N—OR, ═N—NRR′, —C(═NR)—NRR′, —NR—NRR′,—(R)N—C(═NR)—NRR′, O←NR—, >C═NR, —(CH₂)_(n)—NR—COR′, —(CH₂)_(n)—CO—NRR′,—O—(CH₂)_(n)—OR, —O—(CH₂)_(n)—O—(CH₂)_(n)—R, —PRR′, —OPO₃RR′, —PO₂HR and—PO₃R″R″, wherein n is an integer of 1 to 10 and R and R′ eachindependently is H, hydrocarbyl or heterocyclyl, or R and R′ togetherwith the N atom to which they are attached form a 3-7 membered saturatedring optionally containing a further heteroatom selected from O, S andN, wherein the further N atom may be substituted, and R″ is H, a cation,hydrocarbyl or heterocyclyl;

(d) C₁-C₂₅ hydrocarbyl substituted by one or more functional groupsselected from the group consisting of positively charged groups,negatively charged groups, basic groups that are converted to positivelycharged groups under physiological conditions, and acidic groups thatare converted to negatively charged groups under physiologicalconditions;

(e) C₁-C₂₅ hydrocarbyl containing one or more heteroatoms and/or one ormore carbocyclic or heterocyclic moieties;

(f) C₁-C₂₅ hydrocarbyl containing one or more heteroatoms and/or one ormore carbocyclic or heterocyclic moieties and substituted by one or morefunctional groups as defined in (c) and (d) above;

(g) C₁-C₂₅ hydrocarbyl substituted by a residue of an amino acid, apeptide, a protein, a monosaccharide, an oligosaccharide, or apolysaccharide; or

(h) a residue of an amino acid, a peptide, a protein, a monosaccharide,an oligosaccharide, or a polysaccharide;

R₇ may further be —NRR′, wherein R and R′ each is H or C₁-C₂₅hydrocarbyl, optionally substituted by a negatively charged group,preferably SO₃ ⁻;

R₈ may further be H⁺ or a cation R⁺ ₁₀ when R₁, R′₂ and R₆ eachindependently is Y—R₈;

R+₁₀ is a metal cation, an ammonium group or an organic cation;

A⁻ is a physiologically acceptable anion;

m is 0 or 1;

the dotted line at positions 7-8 represents an optional double bond; and

pharmaceutically acceptable salts and optical isomers thereof.

In certain embodiments, the dotted line at positions 7-8 represents adouble bond and the photosensitizer is a chlorophyll of the formula I,II or III.

The pentacyclic chlorophyll compound of formula I wherein M is Mg, R₁ atposition 17³ is phytyloxy, R₂ at position 13² is COOCH₃, R₃ at position13² is an H atom, R₅ is O, R₄ at position 3 is vinyl, the dotted line atpositions 7-8 represents a double bond, R′₄ is methyl or formyl atposition 7 and R₄ is ethyl at position 8, are chlorophyll a and b,respectively, and their derivatives will have a different metal atomand/or different substituents R₁, R₂, R₃, R₄, R′₄ and/or R₅.

The tetracyclic compound of formula II (three pyrroles and one pyrrolinecoupled through four methine linkages) wherein M is absent, R₁ atposition 17³ is a propionic acid, R′₂ at position 15 is —CH₂COOH, R₆ atposition 13² is —COOH, R₄ at position 3 is vinyl, the dotted line atpositions 7-8 represents a double bond, R′₄ is methyl at position 7 andR₄ is ethyl at position 8, is chlorin, and its derivatives will havedifferent metal atoms and/or different substituents R₁, R′₂, R₄, R′₄and/or R₆.

The pentacyclic compound of formula III wherein M is absent, X isoxygen, R₁ at position 17³ is a propionic acid, R₄ at position 3 isvinyl, the dotted line at positions 7-8 represents a double bond, R′₄ ismethyl at position 7 and R₄ is ethyl at position 8 is purpurin-18, andits derivatives will have different metal atoms and/or differentsubstituents R₁, R₄, R′₄, and/or X other than oxygen.

In certain other embodiments, the positions 7-8 are hydrogenated and thephotosensitizer is a bacteriochlorophyll of the formula I, II or III.The pentacyclic compounds of formula I wherein M is Mg, R₁ at position17³ is phytyloxy or geranylgeranyloxy, R₂ at position 13² is COOCH₃, R₃at position 13² is an H atom, R₅ is O, R₄ at position 3 is acetyl and atposition 8 is ethyl, and the dotted line at positions 7-8 is absent, isbacteriochlorophyll a (Bchla), and its optional derivatives will havedifferent metal atoms and/or different substituents R₁, R₂, R₃, R₄, R′₄and/or R₅.

The tetracyclic (two pyrroles and two pyrroline coupled through fourmethine linkages) compounds of formula II wherein M is absent, methylgroups at positions 2, 7, 12, 18, R₁ at position 17³ is propionic acid,R′₂ at position 13 is COOCH₃, R₄ at position 3 is acetyl and at position8 is ethyl, and the dotted line at positions 7-8 is absent, isbacteriochlorin a. When a vinyl group is attached at positions 3, thecompound is rhodobacteriochlorin ((3¹-vinyl)-bacteriochlorin a).Derivatives of bacteriochlorin a and rhodobacteriochlorin will havedifferent metal atoms and/or different substituents R₁, R′₂, R₄ atposition 3 and/or R₆.

The pentacyclic compound of formula III wherein M is absent, X isoxygen, R₁ at position 17³ is a propionic acid, R₄ at position 3 isacetyl and at position 8 is ethyl, the dotted line at positions 7-8 isabsent, is bacteriopurpurin-18, and its derivatives will have differentmetal atoms and/or different substituents R₁, R₄, R′₄ and/or X otherthan oxygen.

As used herein, the term “hydrocarbyl” means any straight or branched,saturated or unsaturated, acyclic or cyclic, including aromatic,hydrocarbyl radicals, of 1-25 carbon atoms, preferably of 1 to 20 or 1to 10, more preferably 1 to 6, most preferably 2-3 carbon atoms. Thehydrocarbyl may be a lower alkyl radical of 1-6, preferably of 1-4carbon atoms, e.g. methyl, ethyl, propyl, isopropyl, butyl, isobutyl,tert-butyl, or alkenyl, alkynyl or cycloalkyl, or at the position 17 ofthe compounds of formula I, II or III, the hydrocarbyl is a radicalderived from natural Chl and Bchl compounds, e.g. geranylgeranyl(2,6-dimethyl-2,6-octadienyl) or phytyl(2,6,10,14-tetramethyl-hexadec-14-en-16-yl).

In one embodiment, the alkyl group has 10 carbon atoms or more, e.g.—C₁₀H₂₁, —C₁₅H₃₁, —C₁₆H₃₃, —C₁₇H₃₅, —C₁₈H₃₇, —C₂₀H₄₁, and the like.

In another embodiment, the C₁-C₂₅ hydrocarbyl is a straight or branchedC₂-C₂₅ alkenyl or alkynyl radical, preferably of 2-6 carbon atoms, e.g.vinyl, prop-2-en-1-yl, but-3-en-1-yl, pent-4-en-1-yl, hex-5-en-1-yl,ethynyl, propargyl, and the like.

In yet another embodiment, the C₁-C₂₅ hydrocarbyl is a C₃-C₂₅ monocyclicor polycyclic cycloalkyl or partially unsaturated cycloalkyl, preferablyC₃-C₁₄, more preferably C₃-C₇ cycloalkyl, such as cyclopropyl,cyclobutyl, cyclopentyl, cyclohexyl and cycloheptyl.

The hydrocarbyl may further be aryl or aralkyl, wherein the term “aryl”as used herein refers to a “C₆-C₁₄” aromatic carbocyclic group having 6to 14 carbon atoms, preferably 6 to 10 carbon atoms, consisting of asingle, bicyclic or tricyclic ring system, such as phenyl, naphthyl,carbazolyl, anthryl, phenanthryl and the like, and the term “aralkyl”refers to a radical derived from an arylalkyl compound wherein the arylmoiety is preferably a C₆-C₁₄, more preferably a C₆-C₁₀ aryl such asbenzyl, phenanthryl and the like.

The term “heterocyclic ring” or “heterocyclyl” means a radical derivedfrom a saturated, partially unsaturated, optionally substituted,monocyclic, bicyclic or tricyclic heterocycle of 3-12, preferably 5-10,more preferably 5-6 members in the ring containing 1 to 3 heteroatomsselected from O, S and/or N. Particular examples are dihydrofuryl,tetrahydrofuryl, pyrrolynyl, pyrrolydinyl, dihydrothienyl,dihydropyridyl, piperidinyl, quinolinyl, piperazinyl, morpholino or1,3-dioxanyl.

The terms “heteroaryl” or “heteroaromatic moiety” refer to a mono- orpolycyclic heteroaromatic ring that may comprise both carbocyclic andheterocyclic rings, containing 1 to 3 heteroatoms selected from O, Sand/or N and optionally substituted. Particular examples are, withoutbeing limited to, pyrrolyl, furyl, thienyl, pyrazolyl, imidazolyl,oxazolyl, thiazolyl, pyridyl, quinolinyl, isoquinolinyl, pyridazinyl,pyrimidinyl, pyrazinyl, 1,3,4-triazinyl, 1,2,3-triazinyl,1,3,5-triazinyl, benzofuryl, isobenzofuryl, indolyl,imidazo[1,2-a]pyridyl, benzimidazolyl, benzthiazolyl, benzoxazolyl,benzodiazepinyl, and other radicals derived from further polycyclicheteroaromatic rings.

Any “carbocyclyl”, “heterocyclyl”, “aryl” or “heteroaryl” may besubstituted by one or more radicals such as halogen, C₆-C₁₄ aryl, C₁-C₂₅alkyl, nitro, OR, SR, —COR, —COOR, COOR″, —SO₃R, —SO₃R″, —SO₂R, —NHSO₂R,—NRR′, —(CH₂)_(n)—NR—COR′, and —(CH₂)_(n)—CO—NRR′, wherein n, R, R′ andR″ are as defined above. It is to be understood that when a polycyclicheteroaromatic ring is substituted, the substitutions may be in any ofthe carbocyclic and/or heterocyclic rings.

The term “alkoxy” as used herein refers to a group (C₁-C₂₅)alkyl-O—,wherein C₁-C₂₅ alkyl is as defined above. Examples of alkoxy aremethoxy, ethoxy, n-propoxy, isopropoxy, butoxy, isobutoxy, tert-butoxy,pentoxy, hexoxy, —OC₁₂H₂₅, —OC₁₅H₃₁, —OC₁₆H₃₃, —OC₁₇H₃₅, —OC₁₈H₃₇, andthe like. The term “aryloxy” as used herein refers to a group(C₆-C₁₈)aryl-O—, wherein C₆-C₁₈ aryl is as defined above, for example,phenoxy and naphthoxy.

The term “halogen”, as used herein, refers to fluoro, chloro, bromo oriodo.

The hydrocarbon chain of R₇, R₈, R₉, R′₉ and/or R″₉ may optionallycontain one or more heteroatoms such as O, S and/or NH, and/or one ormore carbocyclic rings or heterocyclic ring moieties, wherein“carbocyclic” as used herein encompasses cycloalkyl and aryl as definedherein. In one embodiment, the hydrocarbyl chain contains one or more Oatoms and has a OH end group as represented by an oligooxyethyleneglycolresidue of 4 to 10 carbon atoms, preferably pentaoxyethyleneglycol. Inother embodiments, the hydrocarbyl contains phenyl or pyridyl.

R₇, R₈, R₉, R′₉ and/or R″₉ may also be hydrocarbyl substituted by one ormore functional groups such as halogen, for example Cl, Br, F or I,nitro, oxo, alkoxy (OR), SR, epoxy, epithio, —CONRR′, —COR, COOR″, COSR,—OSO₃R, —SO₃R″, —SO₂R, —NHSO₂R, —SO₂NRR′—NRR′, ═N—OR, ═N—NRR′,—C(═NR)—NRR′, —NR—NRR′, —(R)N—C(═NR)—NRR′, O←NR—, >C═NR,—(CH₂)_(n)—NR—COR′, —(CH₂)_(n)—CO—NRR′, —O—(CH₂)_(n)—OR,—O—(CH₂)_(n)—O—(CH₂)_(n)—R, —PRR′, —OPO₃RR′, —PO₂HR and —PO₃R″R″,wherein n is an integer of 1 to 10 and R and R′ each independently is H,hydrocarbyl, or heterocyclyl, or R and R′ together with the N atom towhich they are attached form a 3-7 membered saturated ring, optionallycontaining one or more heteroatoms selected from the group consisting ofO, S or N, and optionally further substituted at the additional N atom,and R″ is H, a cation, hydrocarbyl, or heterocyclyl.

In certain embodiments, some of the functional groups above are acidicgroups that may convert to negatively charged groups under physiologicalpH, for example COOH, COSH, SO₃H, PO₃H₂, or the functional groups arenegatively charged group such as COO⁻, COS⁻, SO₃ ⁻, or PO₃ ²⁻. Thenegatively charged group and the acidic group may be an end group or agroup within the hydrocarbyl chain. In most preferred embodiments, thehydrocarbyl has 2 or 3 carbon atoms and an end group selected from COO⁻,PO₃ ²⁻, or, most preferably, SO₃ ⁻.

As used herein, “physiological conditions” refers to the conditions indifferent tissues and cell compartments of the body.

In certain embodiments, R₇, R₈, R₉, R′₉ and/or R″₉ may by substituted byat least one positively charged group and/or at least one basic groupthat is converted to a positively charged group under physiologicalconditions. In a preferred embodiment, the at least one positivelycharged group may be a cation derived from a N-containing group such as,but not limited to, an ammonium —N⁺(RR′R″), hydrazinium —(R)N—N⁺(R′R″),ammoniumoxy O←N⁺(RR′)—, iminium >C═N⁺(RR′), amidinium —C(═RN)—N⁺R′R″ orguanidinium —(R)N—C(═NR)—N⁺R′R″ group, wherein R, R′ and R″ are asdefined above. It is to be understood that the positively chargedN-containing group may be an end group, a group within the hydrocarbylchain, or part of a saturated ring in which the N is protonated, asdefined hereinafter. In addition, the at least one positively chargedgroup may also be a cation derived from a N-containing heteroaromaticradical, as defined hereinafter.

In one preferred embodiment, the hydrocarbyl chain is substituted by anammonium group of the formula —N⁺(RR′R″), wherein each of R, R′ and R″independently is H, hydrocarbyl, preferably C₁-C₂₅ alkyl, morepreferably C₁-C₁₀ or C₁-C₆ alkyl, or heterocyclyl. When one of R, R′ orR″ is OH, the group is a hydroxyl ammonium group. Preferably, theammonium group is a quaternary ammonium group wherein R, R′ and R″ eachindependently is C₁-C₆ alkyl such as methyl, ethyl, propyl, butyl,pentyl or hexyl.

In certain embodiments, the ammonium group of the formula —N⁺(RR′R″) isa cyclic group, wherein two of R, R′ and R″ together with the N atomform a 3-7 membered saturated ring, optionally containing a furtherheteroatom selected from the group consisting of O, S and N atom, andoptionally further substituted at the additional N atom, as definedhereinafter. Examples of such cyclic ammonium groups includeaziridinium, pyrrolidinium, piperidinium, piperazinium, morpholinium,thiomorpholinium, azepinium, and the like.

In certain embodiments, the positively charged group is a cation derivedfrom a N-heteroaromatic compound that may be a mono- or polycycliccompound that may further contain O, S or additional N atoms. The ringfrom which the cation is derived should contain at least one N atom andbe aromatic, but the other ring(s), if any, can be partially saturated.Examples of N-heteroaromatic cations include pyrazolium, imidazolium,oxazolium, thiazolium, pyridinium, pyrimidinium, quinolinium,isoquinolinium, 1,2,4-triazinium, 1,3,5-triazinium and purinium.

The cation may also be an onium group not containing N such as, but notlimited to, a phosphonium [—P⁺(RR′R″)], arsonium [—As⁺(RR′R″)], oxonium[—O⁺(RR′)], sulfonium [—S⁺(RR′)], selenonium [—Se⁺(RR′)], telluronium[—Te⁺(RR′)], stibonium [—Sb⁺(RR′R″)], or bismuthonium [—Bi⁺(RR′R″)]group, wherein R, R′ and R″ are as defined above. In preferredembodiments, R, R′ and R″ are H, C₁-C₆ alkyl such as methyl, ethyl,propyl, isopropyl, n-butyl, isobutyl, sec-butyl, pentyl or hexyl, anaryl group, preferably, phenyl, or an aralkyl group, such as benzyl andphenethyl.

In certain other embodiments, R₇, R₈, R₉, R′₉ and/or R″₉ are substitutedby at least one basic group that is converted to a positively chargedgroup under physiological conditions. As defined herein, “a basic groupthat is converted to a positively charged group under physiologicalconditions” is, at least theoretically, any basic group that willgenerate under physiological conditions a positively charged group asdefined herein, wherein the physiological conditions, as used herein, donot refer solely to the serum, but to different tissues and cellcompartments in the body.

In certain embodiments, the basic group is a N-containing group.Examples of such N-containing basic groups include, without beinglimited to, any amino group that will generate an ammonium group, anyimine group that will generate an iminium group, any hydrazine groupthat will generate a hydrazinium group, any aminooxy group that willgenerate an ammoniumoxy group, any amidine group that will generate anamidinium group, any guanidine group that will generate a guanidiniumgroup, all as defined herein. Other examples include phosphino andmercapto groups.

Thus, R₇, R₈, R₉, R′₉ and/or R″₉ may be substituted by at least onebasic group such as —NRR′, —C(═NR)—NR′R″, —NR—NR′R″, —(R)N—C(═NR)—NR′R″,O←NR—, or >C═NR, wherein R, R′ and R″ are as defined above butpreferably C₁-C₂₅ alkyl, more preferably C₁-C₁₀ or C₁-C₆ alkyl, orheterocyclyl. In preferred embodiments, the basic group is an aminogroup NRR′, an end group or a group within the hydrocarbyl chain, thatmay be a secondary amino, wherein only one of R and R′ is H, or atertiary amino wherein none of R and R′ is H, or it may be a cyclicamino wherein R and R′ together with the N atom form a 3-7 memberedsaturated ring, optionally containing a further heteroatom selected fromthe group consisting of O, S and N atom, and optionally furthersubstituted at the additional N atom.

The C₁-C₂₅ hydrocarbyl defined for R₇, R₈, R₉, R′₉ and/or R″₉ may alsobe substituted by the residue of a mono-, oligo- or polysaccharide suchas glycosyl, or of an amino acid, peptide or protein. In addition, R₈,R₉, R′₉ and R″₉ each may independently be is a moiety of anoligosaccharide, or a polysaccharide, preferably a monoaccharide such asglucosamine, and/or the residue of an amino acid, a peptide or aprotein. In one preferred embodiment, R₈, R₉, R′₉ or R″₉ at any of thepositions, but preferably at position 17³, is the residue of an aminoacid, a peptide or a protein. The amino acid, peptide or protein may benegatively charged if they contain a free terminal carboxyl group and/ora residue of an amino acid containing a non-terminal free carboxylicgroup, e.g. aspartic or glutamic acid.

In one embodiment, R₇, R₈, R₉, R′₉ or R″₉ is the residue of an aminoacid containing a hydroxy group, such as serine, threonine and tyrosineor a derivative thereof selected from esters such as alkyl, preferablymethyl, esters, and N-protected derivatives wherein the N-protectinggroup is for example tert-butyloxy, carbobenzoxy or trityl, or peptides(oligopeptide or polypeptide) containing such amino acid and/or aminoacid derivatives. The hydroxylated amino acid or peptide is linked tothe COO⁻ group, preferably at position 17³, of the (bacterio)chlorophyllderivative through its hydroxy group. Examples of such amino acidderivatives are serine methyl ester, N-tert-butyloxycarbonyl-serine,N-trityl-serine methyl ester, tyrosine methyl ester, andN-tert-butoxy-tyrosine methyl ester, and an example of a peptidecontaining said amino acid derivative is N-carbobenzoxy-seryl serinemethyl ester, all of which are prepared as described in the EP 0584552incorporated herein by reference as if fully disclosed herein.

In certain embodiments, R₇, R₈, R₉, R′₉ or R″₉ is the residue of acell-specific or tissue-specific ligand selected from peptides andproteins, which are exemplified by, but not limited to, hormone peptidesand antibodies, e.g. immunoglobulins. The peptide or protein may belinked directly to the —CO group via an ester, thioester or amide bond,or it may be linked via an ester or amide bond to an end functionalgroup of the C₁-C₂₅ hydrocarbyl radical selected from OH, COOH and NH₂.

In certain embodiments, the (bacterio)chlorophyll derivatives used inaccordance with the invention contain COOH, COSH, COO⁻, and/or COS⁻group derived from R₁, R′₂ and R₆ being OH or SH, O⁻R₁₀ ⁺ or S⁻R₁₀ ⁺,respectively, i.e., when a carboxylic or thiocarboxylic group or acarboxylate or thiocarboxylate anion is present at the position 13¹, 15¹(m is 0), 15² (m is 1), and/or 17³.

In certain embodiments R₁, R′₂ and/or R₆ is a basic group —NR₉R′₉ or anammonium group —N⁺R₉R′₉R″₉, or R₅ is N—R₉ or N⁺R₉R′₉.

In certain embodiments, R₁, R′₂ and/or R₆ is Y—R₈ wherein Y is O and R₈is a residue of an amino acid or peptide (oligo or polypeptide) linkedthrough an amide bond via a free —NH₂ group.

The cation R₁₀ ⁺ may be a monovalent or divalent cation derived from analkaline or alkaline earth metal such as K⁺, Na⁺, Li⁺, Ca⁺, morepreferably K⁺; or R₁₀ ⁺ is an organic cation such as herein defined for“a cation derived from a N-containing group”. Preferably, the organiccation is derived from an amine, e.g., NH₄ ⁺.

As defined herein, A⁻ is a physiologically acceptable anion such aschloride, bromide, iodide, perchlorate, sulfate, phosphate or an organicanion such as acetate, benzoate, caprylate, citrate, lactate, malonate,mandelate, mesylate, oxalate, propionate, succinate, tosylate, and thelike.

As defined herein, “a 3-7 membered saturated ring” formed by R and R′together with the N atom to which they are attached may be a ringcontaining only N such as aziridine, pyrrolidine, piperidine, piperazineor azepine, or it may contain a further heteroatom selected from O and Ssuch as morpholine or thiomorpholine. The further N atom in thepiperazine ring may be optionally substituted by alkyl, e.g. C₁-C₆alkyl, that may be substituted by halogen, OH or amino. The onium groupsderived from said saturated rings include aziridinium, pyrrolidinium,piperidinium, piperazinium, morpholinium, thiomorpholinium andazepinium.

In certain embodiments, the (bacterio)chlorophyll derivative used inaccordance with the present invention is unmetalated, namely M is 2H. Inpreferred embodiments, the photosensitizer is metalated and M is Mg, Pd,Pt, Zn, In, Gd, or Yb, more preferably Pd.

In one preferred embodiment, the ophthalmic composition provided by thepresent invention for treatment of eye diseases and disorders associatedwith corneal thinning or scleral stretching comprises chlorophyll orbacteriochlorophyll derivative of the formula I wherein: M representsdivalent Pd; R₁ is —NH—(CH₂)_(n)—SO₃ ⁻R₁₀ ⁺, —NH—(CH₂)_(n)—COO⁻R₁₀ ⁺; or—NH—(CH₂)_(n)—PO₃ ²⁻(R₈ ⁺)₂; R₂ is methoxy; R₃ is —C(═O)—CH₃; R₅ is O;R₁₀ ⁺ is a monovalent cation such as K⁺, Na⁺, Li⁺, or NH₄ ⁺; and n is aninteger from 1 to 10, preferably 2 or 3. Preferably, according to thisembodiment, R₁ is —NH—(CH₂)₃—SO₃ ⁻K⁺.

In more preferred embodiments, the photosensitizer is abacteriochlorophyll of the formula I having a sole negatively chargedgroup (SO₃ ⁻) at position 17, represented by the compound palladiumbacteriopheophorbide a 17³-(3-sulfopropyl) amide potassium salt.

In certain embodiments, the ophthalmic composition for treatment ofcorneal thinning or scleral stretching comprises a derivative ofchlorophyll or bacteriochlorophyll of formula II in which R₆ or both R₁and R₆ are —NR₉R′₉. Preferably according to these embodiments, R₉ is Hand R′₉ is a C₁-C₁₀ alkyl substituted by at least one group selectedfrom a positively charged group, a negatively charged group, an acidicgroup that is converted to a negatively charged group underphysiological conditions, or a basic group that is converted to apositively charged group under physiological conditions. In moreparticular embodiments R′₉ is a C₁-C₆ alkyl substituted by the acidicSO₃H group or an alkaline salt thereof, or by a basic group —NRR′ or—NH—(CH₂)₂₋₆—NRR′, wherein each of R and R′ independently is H, C₁-C₆alkyl optionally substituted by NH₂, or R and R′ together with the Natom form a 5-6 membered saturated ring, optionally containing an O or Natom and optionally further substituted at the additional N atom by—(CH₂)₂₋₆—NH₂.

In certain embodiments, the ophthalmic composition of the inventioncomprises a bacteriochlorophyll derivative of formula II, wherein:

M is 2H, Mg, Pd, or Zn;

R₁ is selected from:

(i) —O⁻R₁₀ ⁺;

(ii) Y—R₈ wherein Y is O or S and R₈ is the residue of an amino acid, apeptide or a protein;

(iii) —NH—CH₂—CH(OH)—CH₂OH;

(iv) —NH—(CH₂)_(n)—OH;

(v) —NH—CH(OH)—CH₃;

(vi) —NH—(CH₂)_(n)—NR—(CH₂)_(n)—OH;

(vii) glycosylamino; or

(viii) NHR′₉ which is as defined for R₆;

R′₂ is C₁-C₆ alkoxy such as methoxy, ethoxy, propoxy or butoxy, morepreferably methoxy;

R₄ is —C(═O)—CH₃, —CH═N—(CH₂)_(n)—SO₃ ⁻R₁₀ ⁺; —CH═N—(CH₂)_(n)—COO⁻R₁₀ ⁺;—CH═N—(CH₂)_(n)—PO₃ ²⁻(R₁₀ ⁺)₂; —CH₂—NH—(CH₂)_(n)—SO₃ ⁻R₁₀ ⁺;—NH—(CH₂)_(n)—COO⁻R₁₀ ⁺; —NH—(CH₂)_(n)—PO₃ ²⁻(R₁₀ ⁺)₂; or —C(CH₃)═NR₉,preferably C(CH₃)═N—(CH₂)_(n)—NH₂, or —C(CH₃)═N—(CH₂)_(n)—N(R)₃ ⁺A⁻;

R₆ is selected from (i) NHR′₉, selected from:

-   -   (a) —NH—(CH₂)_(n)—SO₃ ⁻R₁₀ ⁺, preferably —NH—(CH₂)₂—SO₃R₁₀ ⁺ or        —NH—(CH₂)₃—SO₃R₁₀ ⁺;    -   (b) —NH—(CH₂)_(n)—COO⁻R₁₀ ⁺;    -   (c) —NH—(CH₂)_(n)—PO₃ ²⁻(R₁₀ ⁺)₂;    -   (d) —NH—(CH₂)_(n)—B:    -   (e)

-   -   (f)

preferably —NH—(CH₂)₂-1-morpholino or —NH—(CH₂)₃-piperazino;

-   -   (g)

-   -   (h) —NH—(CH₂)_(n)—N(R″)—(CH₂)_(n)—NRR′, preferably        —NH—(CH₂)₃—NH—(CH₂)₃—NH₂;    -   (j) —NH—(CH₂)_(n)—NRR′, preferably —NH—CH₂—CH₂—NRR′;

R₁₀ ⁺ is H⁺, or a monovalent cation such as K⁺, Na⁺, Li⁺, or NH₄ ⁺, morepreferably K⁺;

X is O, S or NH;

B is a positively charged group selected from ammonium —N⁺RR′R″,preferably —N(CH₃)₃ ⁺A⁻, guanidinium, sulfonium, phosphonium, arsonium;or B is a basic group that is converted to a positively charged groupunder physiological conditions, selected from amino —NRR′, preferably—NH₂, guanidino, phosphino, or arsino;

m is 1, n is an integer from 1 to 10, preferably 2 or 3; and

A⁻ is a physiologically acceptable anion;

wherein R, R′ and R″ each independently is H or C₁-C₆ alkyl.

In preferred embodiments, the pharmaceutical composition comprisesbacteriochlorin or rhodobacteriochlorin derivative of formula II whereinonly R₆ or both R₁ and R₆ are selected from —NH—(CH₂)₂—SO₃R₁₀ ⁺,—NH—(CH₂)₃—SO₃R₁₀ ⁺, —NH—(CH₂)₃—NH—(CH₂)₃—NH₂, —NH—(CH₂)₂-1-morpholino,or —NH—(CH₂)₃-piperazino-(CH₂)₃—NH₂, and R₁₀ ⁺ is a monovalent cationsuch as K⁺, Na⁺, Li⁺, NH₄ ⁺, preferably K⁺. In certain more preferredembodiments, R₁ and R₆ are both —NH—(CH₂)₂—SO₃R₁₀ ⁺ or —NH—(CH₂)₃—SO₃R₁₀⁺.

In other preferred embodiments, chlorin or (rhodo)bacteriochlorinderivatives of the formula II are used in accordance with the invention,having at least one negatively charged group and M is absent or isdivalent Pd or Zn ion, preferably Pd. In these embodiments, R₁ isselected from —O⁻R₁₀ ⁺, —NH—(CH₂)_(n)—SO₃ ⁻R₁₀+, —NH—(CH₂)_(n)—COO⁻R₁₀ ⁺or —NH—(CH₂)_(n)—PO₃ ²⁻(R₁₀ ⁺)₂, or R₁ is Y—R₈ wherein Y is O, S and R₈is the residue of an amino acid, a peptide or a protein; R′₂ is C₁-C₆alkoxy such as methoxy, ethoxy, propoxy, butoxy, more preferablymethoxy; R₄ is selected from —C(═O)—CH₃, —CH═N—(CH₂)_(n)—SO₃ ⁻R₁₀ ⁺,—CH═N—(CH₂)_(n)—COO⁻R₁₀ ⁺, —CH═N—(CH₂)_(n)—PO₃ ²⁻(R₁₀ ⁺)₂, or—CH₂—NH—(CH₂)_(n)—SO₃ ⁻R₁₀ ⁺; R₆ is —NH—(CH₂)_(n)—SO₃ ⁻R₁₀ ⁺,—NH—(CH₂)_(n)—COO⁻R₁₀ ⁺, or —NH—(CH₂)_(n)—PO₃ ²⁻(R₁₀ ⁺)₂; R₁₀ ⁺ is amonovalent cation such as K⁺, Na⁺, Li⁺, or NH₄ ⁺, more preferably K⁺; mis 1, and n is an integer from 1 to 10, preferably 2 or 3.

In a more preferred embodiment, the negatively charged photosensitizerused in accordance with the invention is a bacteriochlorin derivative ofthe formula II wherein: M is Pd; R₁ is —O⁻R₁₀ ⁺, NH—(CH₂)_(n)—SO₃ ⁻R₁₀⁺, or Y—R₈ wherein Y is O or S and R₈ is the residue of a protein,preferably immunoglobulin; R′₂ is C₁-C₆ alkoxy such as methoxy, ethoxy,propoxy, butoxy, more preferably methoxy; R₄ is —C(═O)—CH₃,—CH═N—(CH₂)_(n)—SO₃ ⁻R₁₀ ⁺ or —CH₂—NH—(CH₂)_(n)—SO₃ ⁻R₁₀ ⁺; R₆ is—NH—(CH₂)_(n)—SO₃ ⁻R₁₀ ⁺, NH—(CH₂)_(n)—COO⁻R₁₀ ⁺, or NH—(CH₂)_(n)—PO₃²⁻(R₈ ⁺)₂; R₁₀ ⁺ is a monovalent cation such as K⁺, Na⁺, Li⁺ or NH₄ ⁺,more preferably K⁺; m is 1, and n is 2 or 3, more preferably 2.

Examples of rhodobacteriochlorin derivatives of formula II having one,two or three negatively charged groups at positions 3, 13 and/or 17 are:

-   Palladium 3¹-oxo-15-methoxycarbonylmethyl-rhodobacteriochlorin 13¹    -(2-sulfoethyl)amide dipotassium salt (herein designated WST11);-   Palladium 3¹-oxo-15-methoxycarbonylmethyl-rhodobacteriochlorin    13¹-(-sulfopropyl)amide dipotassium salt (compound 1);-   Palladium 3¹-oxo-15-methoxycarbonylmethyl-rhodobacteriochlorin    13¹,17³-di-(2-sulfoethyl)amide dipotassium salt (compound 2);-   Palladium 3¹-oxo-15-methoxycarbonylmethyl-rhodobacteriochlorin    13¹,17³-di-(3-sulfopropyl)amide dipotassium salt (compound 3);-   3¹-oxo-15-methoxycarbonylmethyl-rhodobacteriochlorin    13¹-(2-sulfoethyl) amide dipotassium salt (compound 4);-   3¹-oxo-15-methoxycarbonylmethyl-rhodobacteriochlorin    13¹-(-sulfopropyl)amide dipotassium salt (compound 5);-   3¹-oxo-15-methoxycarbonylmethyl-rhodobacteriochlorin    13¹,17³-di-(2-sulfoethyl)amide dipotassium salt (compound 6);-   3¹-oxo-15-methoxycarbonylmethyl-rhodobacteriochlorin    13¹,17³-di-(3-sulfopropyl)amide dipotassium salt (compound 7);-   Zinc 3¹-oxo-15-methoxycarbonylmethyl-rhodobacteriochlorin    13¹-(2-sulfoethyl) amide dipotassium salt;-   Palladium 3¹-oxo-15-methoxycarbonylmethyl-rhodobacteriochlorin    13¹-(2-sulfoethyl)amide, 17³-(N-immunoglobulin G)amide potassium    salt;-   Palladium 3¹-oxo-15-methoxycarbonylmethyl-rhodobacteriochlorin    13¹-(2-carboxyethyl)amide dipotassium salt;-   Palladium 3¹-oxo-15-methoxycarbonylmethyl-rhodobacteriochlorin    13¹-(3-phosphopropyl)amide tripotassium salt-   Palladium    3¹-(3-sulfopropylimino)-15-methoxycarbonylmethyl-rhodobacteriochlorin    13¹,17³-di(3-sulfopropyl)amide tripotassium salt;-   Palladium    3¹-(3-sulfopropylamino)-15-methoxycarbonylmethyl-rhodobacteriochlorin    13¹,17³-di(3-sulfopropyl)amide tripotassium salt.-   Palladium 3¹-oxo-15-methoxycarbonylmethyl-rhodobacteriochlorin    13¹,17³-di(3-propionyl)amide salt;-   Palladium 3¹-oxo-15-methoxycarbonylmethyl-rhodobacteriochlorin    13¹,17³-di[2-(3-propionylamino)-sulfoethyl]amide dipotassium salt;-   Palladium 3¹-oxo-15-methoxycarbonylmethyl-rhodobacteriochlorin    13¹,17³-di[2-(3-propionylamino)-phosphoethyl]amide dipotassium salt;-   Palladium 3¹-oxo-15-methoxycarbonylmethyl-rhodobacteriochlorin    13¹,17³-di(3-thiopropionyl)amide dipotassium salt    3¹-hydroxy-3¹-deoxo-bacteriopheophorbide a;-   3¹-(Pyridin-4-ylmethoxy)-3¹-deoxo-bacteriopheophorbide a;-   Bacteriopheophorbide a    17³-[(2,6-dichloro-4-methoxyphenyl)(2,4-dichlorophenyl)]methyl    ester;-   3¹-Hydroxy-3¹-deoxo-bacteriopheophorbide a    17³-[(2,6-dichloro-4-methoxyphenyl)(2,4-dichlorophenyl)]methyl    ester;-   3¹-trifluoroacetoxy-3¹-deoxo-bacteriopheophorbide a    17³-[(2,6-dichloro-4-methoxyphenyl)(2,4-dichlorophenyl)]methyl    ester;-   3¹-Bromo-3¹-deoxo-bacteriopheophorbide a;-   3-Vinyl-3-deacetyl-bacteriopheophorbide;-   3¹-(2-Hydroxyethoxy)-3¹-deoxo-bacteriopheophorbide a;-   3¹-(2,2,2-Trifluoroethoxy)-3¹-deoxo-bacteriopheophorbide a;-   3¹-(2-Mercaptoethylsulfanyl)-3¹-deoxo-bacteriopheophorbide a;-   3¹-(2-Hydroxyethylamino)-3¹-deoxo-bacteriopheophorbide a;-   Cobalt(III) 3¹-oxo-15-methoxycarbonylmethyl-rhodobacteriochlorin    13¹-(2-sulfoethyl)amide dipotassium salt;-   Iron(III) 3¹-oxo-15-methoxycarbonylmethyl-rhodobacteriochlorin    13¹-(2-sulfoethyl)amide dipotassium salt; and-   Nickel(II) Bacteriopheophorbide a;-   Platinum(II) Bacteriopheophorbide a.

In a preferred embodiment, the compounds used in accordance with thepresent invention are pharmaceutically acceptable salts of taurinated orhomotaurinated bacteriochlorin derivatives with a monovalent or divalentalkaline or alkaline earth metal cation, or with NH₄ ⁺ selected from:

-   Palladium 3¹-oxo-15-methoxycarbonylmethyl-rhodobacteriochlorin    13¹-(2-sulfoethyl)amide;-   Palladium 3¹-oxo-15-methoxycarbonylmethyl-rhodobacteriochlorin    13¹-(3-sulfopropyl)amide;-   Palladium 3¹-oxo-15-methoxycarbonylmethyl-rhodobacteriochlorin    13¹,17³-di-(2-sulfoethyl)amide;-   Palladium 3¹-oxo-15-methoxycarbonylmethyl-rhodobacteriochlorin    13¹,17³-di-(3-sulfopropyl)amide;-   3¹-oxo-15-methoxycarbonylmethyl-rhodobacteriochlorin    13¹-(2-sulfoethyl)amide;-   3¹-oxo-15-methoxycarbonylmethyl-rhodobacteriochlorin    13¹-(-sulfopropyl)amide;-   3¹-oxo-15-methoxycarbonylmethyl-rhodobacteriochlorin    13¹,17³-di-(2-sulfoethyl)amide; and-   3¹-oxo-15-methoxycarbonylmethyl-rhodobacteriochlorin    13¹,17³-di-(3-sulfopropyl)amide.

Most preferred compounds are WST11 and compounds 1-7.

The bacteriochlorophyll derivatives used in the invention can beprepared by the methods described in U.S. Pat. No. 7,947,672 or in WO2005/120573. For the preparation of compounds wherein R₈ is the residueof an amino acid, peptide or protein, the methods described in EP0584552 may be applied. For the preparation of negatively chargedbacteriochlorin derivatives wherein R₈ is a residue of amino acid, themethod disclosed in EP 0584552 may be combined with the method describedin Scheme 1 of U.S. Pat. No. 7,947,672.

Method for the preparation of negatively-charged compounds of formula IIare disclosed in U.S. Pat. No. 7,947,672 mentioned above. For example,preparation of a bacteriochlorin compound wherein R₁ is —O⁻ R₁₀ ⁺; R′₂is —OCH₃; R₃ is acetyl; R₆ is a group —NH—(CH₂)_(n)—SO₃ ⁻R₁₀ ⁺,—NH—(CH₂)_(n)—COO⁻R₁₀ ⁺, or —NH—(CH₂)_(n)—PO₃ ²⁻(R₁₀ ⁺)₂; R₁₀ ⁺ is amonovalent cation; m is 1 and n is 1 to 10, comprises: (i) reacting thecorresponding metalated bacteriopheophorbide (M-bacteriopheophorbide) offormula I herein wherein R₁ is OH with an aminosulfonic acid of theformula H₂N—(CH₂)_(n)—SO₃H (e.g., taurine H₂N—(CH₂)₂—SO₃H or homotaurineH₂N—(CH₂)₃—SO₃H)aminocarboxylic acid of the formula H₂N—(CH₂)_(n)—COOHor aminophosphonic acid of the formula H₂N—(CH₂)_(n)—PO₃H₂,respectively, in a R₁₀ ⁺-buffer; and (ii) isolating the desired compoundof formula II.

Bacteriochlorins of formula II having the same negatively charged groupsmentioned above at positions 13 and 17 may be prepared by reacting thecorresponding M-bacteriopheophorbide with an excess of the reagentaminosulfonic, aminocarboxylic or aminophosphonic acid as describedabove, and isolation of the desired 13,17-disubstituted derivative, or adifferent route can be followed as disclosed in U.S. Pat. No. 7,947,672.For example, a bacteriochlorin of the formula II wherein R₁ and R₆ areeach a group —NH—(CH₂)_(n)—SO₃ ⁻R₁₀ ⁺; R₂ is —OCH₃; R₃ is acetyl; R₁₀ ⁺is a monovalent cation; m is 1 and n is 1 to 10, are prepared by: (i)coupling the corresponding M-bacteriopheophorbide of formula I whereinR₁ is OH with N-hydroxy-sulfosuccinimide (sulfo NHS) in the presence of1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC); (ii) reacting theresulting M-bacteriopheophorbide-17³-N-hydroxysulfosuccinimide esterwith an excess of an aminosulfonic acid of the formulaH₂N—(CH₂)_(n)—SO₃H in a R₈ ⁺-buffer, thus obtaining a compound offormula I having a sole negatively charged group at position 17; (iii)reacting this product with an excess of H₂N—(CH₂)_(n)—SO₃H in a R₈⁺-buffer; and isolating the desired bacteriochlorin of formula II. Whenthe aminosulfonic acid is replaced by aminocarboxylic or aminophosphonicacid, the corresponding carboxylate and phosphonate derivatives areobtained.

In other embodiments, the photosensitizer used in accordance with theinvention is a purpurin-18 or bacteriopurpurin-18 derivative of formulaIII, wherein X is —NR₇, R₇ is —NRR′, R is H and R′ is C₁-C₆ alkylsubstituted by SO₃— or an alkaline salt thereof, preferably thephotosensitizer is a bacteriopurpurin-18, wherein X is —NR₇ and R₇ is—NH—(CH₂)₃—SO₃ ⁻R₁₀ ⁺, wherein R⁺ ₁₀ is a metal cation, an ammoniumgroup or an organic cation, preferably K⁺.

In certain embodiments, the pharmaceutical compositions used inaccordance with the invention comprise a chlorophyll orbacteriochlorophyll derivative of the formula I, II or III containing atleast one positively charged group and/or at least one basic group thatis converted to a positively charged group at the physiological pH,wherein the positively charged groups and basic group are as definedabove.

In certain embodiments, the photosensitizer is a chlorophyll or(rhodo)bacteriochlorophyll derivative of formula II and R₆ is —NR₉R′₉,R₉ is H and R′₉ is HOCH₂—CH(OH)—CH₂—.

In certain preferred embodiments, the pharmaceutical composition usedaccording to the invention comprises a bacteriochlorin orrhodobacteriochlorin derivative of the formula II wherein R₁ is selectedfrom OH, —NR₉R′₉, or —NR₉—CH₂—CH(OH)—CH₂OH; and R₆ is selected from—NR₉R′₉ or —NR₉—CH₂—CH(OH)—CH₂OH, wherein R₉ is H or C₁-C₆ alkyl; andR′₉ is C₁-C₂₅ hydrocarbyl substituted by at least one positively chargedgroup and/or at least one basic group that is converted to a positivelycharged group under physiological conditions. In preferred embodiments,R′₉ is C₁-C₂₅ alkyl, preferably C₁-C₁₀, more preferably C₁-C₆ alkyl,substituted by at least one positively charged group, more preferably—N⁺RR′R″, or by at least one basic group, preferably —NRR′, andoptionally interrupted by a —N(R″)-group, wherein R and R′ eachindependently is H, C₁-C₆ alkyl optionally substituted by NR″R″, orheterocyclyl such as pyridyl, or R and R′ together with the N atom forma 6-membered ring further containing an O, S or N atom, and R″ is H orC₁-C₆ alkyl.

Particular compounds used in accordance with these preferred embodimentsare positively charged bacteriochlorophyll derivatives wherein M isabsent or is Pd; R′₂ is —OR₈ wherein R₈ is C₁-C₆ alkyl; R₄ is —COCH₃;and wherein:

(a) R₁ is OH and R₆ is —NHR′₉;

(b) R₁ and R₆ are both the same —NHR′₉ group;

(c) R₁ is —NH—CH₂—CH(OH)—CH₂OH and R₆ is a —NHR′₉ group;

(d) R₁ is a NHR′₉ group and R₆ is —NH—CH₂—CH(OH)—CH₂OH; and

(e) R₆ is —NH—CH₂—CH₂—NRR′; and R₁ is selected from the group consistingof

-   -   —NH—(CH₂)_(n)—OH;    -   —NH—CH(OH)—CH₃;    -   —NH—(CH₂)_(n)—NR—(CH₂)_(n)—OH; or    -   —glycosylamino.

The NHR′₉ group is selected from:

-   -   (i) —NH—(CH₂)_(n)—NRR′ or —NH—(CH₂)_(n)—N⁺RR′R″;    -   (ii) —NH—(CH₂)_(n)—N(R″)—(CH₂)_(n)—NRR′;    -   (iii)

-   -   (iv)

and

-   -   (v)

wherein

X is O, S or NR;

R, R′ and R″ are as defined above, but preferably each independently isH or C₁-C₆ alkyl, more preferably methyl or ethyl;

n is an integer from 1 to 10, preferably 2 to 6, more preferably 2 or 3;and

m is an integer from 1 to 6, preferably 1 to 3.

-   -   Particular examples of such positively charged compounds or        compounds that become positively charged under physiological pH        are compounds include the herein designated compounds 4′-7′,8-12        and 24-75:

-   Palladium    3¹-oxo-15-methoxycarbonylmethyl-rhodobacteriochlorin-13¹-(2-N³-trimethylammoniumethyl)amide    chloride salt (compound 12);

-   Palladium    3¹-oxo-15-methoxycarbonylmethyl-rhodobacteriochlorin-13¹-(2-N³-(trimethylammoniumethyl)amide    acetate salt (compound 24);

-   Palladium    3¹-oxo-15-methoxycarbonylmethyl-rhodobacteriochlorin-13¹-(2-N²-dimethylaminoethyl)amide    (compound 25);

-   Palladium    3¹-oxo-15-methoxycarbonylmethyl-rhodobacteriochlorin-13¹-(3-N²-dimethylaminopropyl)amide    (compound 26);

-   Palladium    3¹-oxo-15-methoxycarbonylmethyl-rhodobacteriochlorin-13¹-(2-[(2-aminoethyl)amino]ethyl)amide    (compound 27);

-   Palladium    3¹-oxo-15-methoxycarbonylmethyl-rhodobacteriochlorin-13¹-([2-bis(2-aminoethyl)amino]ethyl)amide    (compound 28);

-   Palladium    3¹-oxo-15-methoxycarbonylmethyl-rhodobacteriochlorin-13¹-(2-morpholino-N-ethyl)amide    (compound 29);

-   Palladium    3¹-oxo-15-methoxycarbonylmethyl-rhodobacteriochlorin-13¹-(2-piperazino-N-ethyl)amide    (compound 30);

-   Palladium    3¹-oxo-15-methoxycarbonylmethyl-rhodobacteriochlorin-13¹-(2-[(2-N²-diethylaminoethyl)amino]ethyl)amide    (compound 31);

-   Palladium    3¹-oxo-15-methoxycarbonylmethyl-rhodobacteriochlorin-13¹-(3-[(3-aminopropyl)amino]propyl)amide    (compound 32);

-   3¹-oxo-15-methoxycarbonylmethyl-rhodobacteriochlorin-13¹,17³-di(2-aminoethyl)amide    (compound 4′);

-   3¹-oxo-15-methoxycarbonylmethyl-rhodobacteriochlorin-13¹,17³-di(2-N³-trimethylammoniumethyl)amide    dicitrate salt (compound 5′);

-   3¹-oxo-15-methoxycarbonylmethyl-rhodobacteriochlorin-13¹,17³-di(3-aminopropyl)amide    (compound 6′);

-   3¹-oxo-15-methoxycarbonylmethyl-rhodobacteriochlorin-13¹,17³-di(3-N³-trimethylammoniumpropyl)amide    dicitrate salt (compound 7′);

-   3¹-oxo-15-methoxycarbonylmethyl-rhodobacteriochlorin-13¹,17³-di(6-aminohexyl)amide    (compound 8);

-   3¹-oxo-15-methoxycarbonylmethyl-rhodobacteriochlorin-13¹,17³-di(6-N³-trimethylammoniumhexyl)amide    dicitrate salt (compound 9);

-   Palladium 3¹-oxo-15-methoxycarbonylmethyl-rhodobacteriochlorin    13¹,17³-di(2-aminoethyl)amide (compound 10);

-   Palladium 3¹-oxo-15-methoxycarbonylmethyl-rhodobacteriochlorin    13¹,17³-di(2-N³-trimethylammoniumethyl)amide diphosphate salt    (compound 11);

-   Palladium    3¹-oxo-15-methoxycarbonylmethyl-rhodobacteriochlorin-13¹,17³-di(2-N³-trimethylammoniumethyl)amide    diacetate salt (compound 33);

-   Palladium    3¹-oxo-15-methoxycarbonylmethyl-rhodobacteriochlorin-13¹,17³-di(3-aminopropyl)amide    (compound 34);

-   Palladium    3¹-oxo-15-methoxycarbonylmethyl-rhodobacteriochlorin-13¹,17³-di(4-aminobutyl)amide    (compound 35);

-   Palladium    3¹-oxo-15-methoxycarbonylmethyl-rhodobacteriochlorin-13¹,17³-di(2-N²-dimethylaminoethyl)amide    (compound 36);

-   Palladium    3¹-oxo-15-methoxycarbonylmethyl-rhodobacteriochlorin-13¹,17³-di(3-N²-dimethylaminopropyl)amide    (compound 37);

-   Palladium    3¹-oxo-15-methoxycarbonylmethyl-rhodobacteriochlorin-13¹,17³-di-(2-[(2-aminoethyl)amino]ethyl)amide    (compound 38);

-   Palladium    3¹-oxo-15-methoxycarbonylmethyl-rhodobacteriochlorin-13¹,17³-di-(2-[(2-N²-diethylaminoethyl)amino]ethyl)    amide (compound 39);

-   Palladium    3¹-oxo-15-methoxycarbonylmethyl-rhodobacteriochlorin-13¹,17³-di(2-morpholino-N-ethyl)amide    (compound 40);

-   Palladium    3¹-oxo-15-methoxycarbonylmethyl-rhodobacteriochlorin-13¹,17³-di(2-piperazino-N-ethyl)amide    (compound 41);

-   Palladium    3¹-oxo-15-methoxycarbonylmethyl-rhodobacteriochlorin-13¹,17³-di-(3-[(3-aminopropyl)amino]propyl)amide    (compound 42);

-   Palladium    3¹-oxo-15-methoxycarbonylmethyl-rhodobacteriochlorin-13¹,17³-di([2-bis(2-aminoethyl)amino]ethyl)amide    (compound 43);

-   Palladium    3¹-oxo-15-methoxycarbonylmethyl-rhodobacteriochlorin-13¹,17³-di(2-N-(2′-pyridyl)aminoethyl)amide    (compound 44);

-   Palladium    3¹-oxo-15-methoxycarbonylmethyl-rhodobacteriochlorin-13¹,17³-di(2-N²-diethylaminoethyl)amide    (compound 45);

-   Palladium    3¹-oxo-15-methoxycarbonylmethyl-rhodobacteriochlorin-13¹-(2-aminoethyl)amide-17³-(2,3-dihydroxypropyl)    amide (compound 48);

-   Palladium    3¹-oxo-15-methoxycarbonylmethyl-rhodobacteriochlorin-13¹-(2-N²-dimethylaminoethyl)amide-17³-(2,3-dihydroxypropyl)amide    (compound 50);

-   Palladium    3¹-oxo-15-methoxycarbonylmethyl-rhodobacteriochlorin-13¹-(2-[(2-aminoethyl)amino]ethyl)amide-17³-(2,3-dihydroxypropyl)amide    (compound 55);

-   Palladium    3¹-oxo-15-methoxycarbonylmethyl-rhodobacteriochlorin-13¹-(2-N-(2′-pyridyl)aminoethyl)amide-17³-(2,3-dihydroxypropyl)amide    (compound 57);

-   Palladium    3¹-oxo-15-methoxycarbonylmethyl-rhodobacteriochlorin-13¹-([2-bis(2-aminoethyl)amine]ethyl)amide-17³-(2,3-dihydroxypropyl)amide    (compound 59);

-   Palladium    3¹-oxo-15-methoxycarbonylmethyl-rhodobacteriochlorin-13¹-(3-aminopropyl)amide-17³-(2,3-dihydroxypropyl)amide    (compound 60);

-   Palladium    3¹-oxo-15-methoxycarbonylmethyl-rhodobacteriochlorin-13¹-(4-aminobutyl)amide-17³-(2,3-dihydroxypropyl)    amide (compound 61);

-   Palladium    3¹-oxo-15-methoxycarbonylmethyl-rhodobacteriochlorin-13¹-(2-N²-diethylaminoethyl)amide-17³-(2,3-dihydroxy    propyl)amide (compound 62);

-   Palladium    3¹-oxo-15-methoxycarbonylmethyl-rhodobacteriochlorin-13¹-(2-N-ethylaminoethyl)amide-17³-(2,3-dihydroxy    propyl)amide (compound 63);

-   Palladium    3¹-oxo-15-methoxycarbonylmethyl-rhodobacteriochlorin-13¹-(3-N-methylaminopropyl)amide-17³-(2,3-dihydroxypropyl)amide    (compound 64);

-   Palladium    3¹-oxo-15-methoxycarbonylmethyl-rhodobacteriochlorin-13¹-(3-N-(2′-pyridyl)aminopropyl)amide-17³-(2,3-dihydroxypropyl)amide    (compound 71);

-   Palladium    3¹-oxo-15-methoxycarbonylmethyl-rhodobacteriochlorin-13¹-(4-N-(2′-pyridyl)aminobutyl)amide-17³-(2,3-dihydroxypropyl)amide    (compound 72);

-   Palladium    3¹-oxo-15-methoxycarbonylmethyl-rhodobacteriochlorin-13¹-(2,3-dihydroxypropyl)amide-17³-(2-trimethylammoniumethyl)amide    (compound 46);

-   Palladium    3¹-oxo-15-methoxycarbonylmethyl-rhodobacteriochlorin-13¹-(2,3-dihydroxypropyl)amide-17³-(2-aminoethyl)    amide (compound 47);

-   Palladium    3¹-oxo-15-methoxycarbonylmethyl-rhodobacteriochlorin-13¹-(2,3-dihydroxypropyl)amide-17³-(2-N²-dimethyl    aminoethyl)amide (compound 49);

-   Palladium    3¹-oxo-15-methoxycarbonylmethyl-rhodobacteriochlorin-13¹-(2,3-dihydroxypropyl)amide-17³-(2-[(2-aminoethyl)amino]ethyl)amide    (compound 51);

-   Palladium    3¹-oxo-15-methoxycarbonylmethyl-rhodobacteriochlorin-13¹-(2,3-dihydroxypropyl)amide-17³-(2-[(2-N²-diethyl    aminoethyl)amino]ethyl)amide (compound 52);

-   Palladium    3¹-oxo-15-methoxycarbonylmethyl-rhodobacteriochlorin-13¹-(2,3-dihydroxypropyl)amide-17³-(2-morpholino-N-ethyl)amide    (compound 53);

-   Palladium    3¹-oxo-15-methoxycarbonylmethyl-rhodobacteriochlorin-13¹-(2,3-dihydroxypropyl)amide-17³-(2-piperazino-N-ethyl)amide    (compound 54);

-   Palladium    3¹-oxo-15-methoxycarbonylmethyl-rhodobacteriochlorin-13¹-(2,3-dihydroxypropyl)amide-17³-(2-N-(2′-pyridyl)aminoethyl)amide    (compound 56);

-   Palladium    3¹-oxo-15-methoxycarbonylmethyl-rhodobacteriochlorin-13¹-(2,3-dihydroxypropyl)amide-17³-([2-bis(2-aminoethyl)amino]ethyl)amide    (compound 58);

-   Palladium    3¹-oxo-15-methoxycarbonylmethyl-rhodobacteriochlorin-13¹-(2,3-dihydroxypropyl)amide-17³-(3-N-(2′-pyridyl)aminopropyl)amide    (compound 73);

-   Palladium    3¹-oxo-15-methoxycarbonylmethyl-rhodobacteriochlorin-13¹-(2,3-dihydroxypropyl)amide-17³-(4-N-(2′-pyridyl)aminobutyl)amide    (compound 74);

-   Palladium    3¹-oxo-15-methoxycarbonylmethyl-rhodobacteriochlorin-13¹-(2-N²-dimethylaminoethyl)amide-17³-(2-hydroxy    ethyl)amide (compound 65);

-   Palladium    3¹-oxo-15-methoxycarbonylmethyl-rhodobacteriochlorin-13¹-(2-N²-dimethylaminoethyl)amide-17³-(3-hydroxy    propyl)amide (compound 66);

-   Palladium    3¹-oxo-15-methoxycarbonylmethyl-rhodobacteriochlorin-13¹-(2-N²-dimethylaminoethyl)amide-17³-(2-hydroxy    propyl)amide (compound 67);

-   Palladium    3¹-oxo-15-methoxycarbonylmethyl-rhodobacteriochlorin-13¹-(2-N²-dimethylaminoethyl)amide-17³-((R)-2-hydroxypropyl)amide    (compound 68);

-   Palladium    3¹-oxo-15-methoxycarbonylmethyl-rhodobacteriochlorin-13¹-(2-N²-dimethylaminoethyl)amide-17³-((S)-2-hydroxypropyl)amide    (compound 69);

-   Palladium    3¹-oxo-15-methoxycarbonylmethyl-Rhodobacteriochlorin-13¹-(2-N²-dimethylaminoethyl)amide-17³-(2-(2-hydroxyethyl    amino)ethyl) amide (compound 70); and

-   Palladium    3¹-oxo-15-methoxycarbonylmethyl-rhodobacteriochlorin-13¹-(2-N²-dimethylaminoethyl)amide-17³-(glycosyl)    amide (compound 75).

The compounds listed above can be prepared e.g., as described in detailin WO 2005/120573 mentioned above.

Other positively charged bacteriochlorin derivatives and basicbacteriochlorin derivatives that become positively charged underphysiological pH are compounds of the formula II, wherein M is Pd, R′₂is —OR₈ wherein R₈ is C₁-C₆ alkyl, preferably methyl, R₄ is —COCH₃, andR₁ and/or R₆ are —NR₉R′₉, wherein R₉ is H and R′₉ is C₁-C₂₅ hydrocarbyl,preferably C₁-C₂₅ alkyl, more preferably C₁-C₁₀ alkyl, substituted by:(i) a guanidino or guanidinium group; (ii) a sulfonium group; (iii) aphosphino or phosphonium group; (iv) an arsino or arsonium group.

In a more preferred embodiment, R₁ and R₆ are both the same groupselected from: (i) —NH—(CH₂)_(n)—C(═NH)—NH₂ or—NH—(CH₂)_(n)—C(═NH)—N⁺(R)₃A⁻, more preferably,—NH—(CH₂)_(n)—C(═NH)—N(CH₃)₃ ⁺A⁻;

(ii) —NH—(CH₂)_(n)—S⁺(R)₂ A⁻, more preferably, —NH—(CH₂)_(n)—S(CH₃)₂⁺A⁻;

(iii) —NH—(CH₂)_(n)—P(R)₂, more preferably, —NH—(CH₂)_(n)—P(CH₃)₂ orNH—(CH₂)_(n)—P⁺(R)₃ A⁻, more preferably, —NH—(CH₂)_(n)—P⁺(CH₃)₃ A⁻; or

(iv) —NH—(CH₂)_(n)—As(R)₂, more preferably, —NH—(CH₂)_(n)—As(CH₃)₂ orNH—(CH₂)_(n)—As⁺(R)₃ A⁻, more preferably, —NH—(CH₂)_(n)—As⁺(CH₃)₃ A⁻,wherein n is an integer from 1 to 10, preferably 2, 3 or 6.

Examples of such compounds are the compounds herein designated compounds14, 14a, 15-19:

-   Palladium 3¹-oxo-15-methoxycarbonylmethyl-rhodobacteriochlorin    13¹,17³-di(2-guanidinoethyl)amide (compound 14);-   Palladium 3¹-oxo-15-methoxycarbonylmethyl-rhodobacteriochlorin    13¹,17³-di(2-trimethylguanidiniumethyl)amide (compound 14a);-   Palladium    3¹-oxo-15-methoxycarbonylmethyl-rhodobacteriochlorin-13¹-(2-S²-dimethylsulfoniumethyl)amide    citrate salt (compound 15);-   3¹-oxo-15-methoxycarbomylmethyl-rhodobacteriochlorin-13¹,17³-di(2-P³-trimethylphosphoniumethyl)amide    dicitrate salt (compound 17);-   3¹-Oxo-15-methoxycarbonylmethyl-rhodobacteriochlorin-13¹,17³-di(2-dimethylphosphinoethyl)amide    (compound 18); and-   3¹-Oxo-15-methoxycarbonylmethyl-rhodobacteriochlorin-13¹,17³-di(2-As³-trimethylarsoniumethyl)amide    dicitrate salt (compound 19).

The above compounds may be prepared as taught in WO 2005/120573.

In still further preferred embodiments, the Bchl derivative used inaccordance with the invention is a bacteriochlorin derivative of theformula II, wherein M is 2H or Pd, R′₂ is —OR₈ wherein R₈ is C₁-C₆alkyl, preferably methyl, R₄ is —C(CH₃)═NR₉, and R₁ and/or R₆ are—NR′₉R″₉, wherein R′₉ is H and R₉ and R″₉ are C₁-C₂₅ hydrocarbyl,preferably C₁-C₂₅ alkyl, more preferably C₁-C₁₀ alkyl, substituted by atleast one amino end group or a positively charged group, more preferablyan ammonium end group of the formula —N⁺(RR′R″)A⁻, wherein R, R′ and R″are preferably the same C₁-C₆ alkyl, preferably methyl, and A⁻ is ananion. In a more preferred embodiment of the invention, R₄ is a group ofthe formula —C(CH₃)═N—(CH₂)_(n)—NH₂ or —C(CH₃)═N—(CH₂)_(n)—N(R)₃ ⁺A⁻,most preferably —C(CH₃)═N—(CH₂)_(n)—N(CH₃)₃ ⁺A⁻, and R₁ and R₆ are agroup of the formula —NH—(CH₂)_(n)—NH₂ or NH—(CH₂)_(n)—N(R)₃ ⁺A⁻, morepreferably, —NH—(CH₂)_(n)—N(CH₃)₃ ⁺A⁻, wherein n is an integer from 1 to10, but preferably 2, 3 or 6. Examples of such compounds are the hereindesignated compounds 20-23:

-   3¹-(aminoethylimino)-15-methoxycarbonylmethyl-rhodobacteriochlorin-13¹,17³-di(2-aminoethyl)amide    (compound 20);-   Palladium    3¹-(aminoethylimino)-15-methoxycarbonylmethyl-rhodobacteriochlorin    13¹,17³-di(2-aminoethyl)amide (compound 21);-   3¹-(trimethylammoniumethylimino)-15-methoxycarbonylmethyl-rhodobacteriochlorin    13¹,17³-di(2-trimethylammoniumethyl)amide (compound 22); and-   Palladium    3¹-(trimethylammoniumethylimino)-15-methoxycarbonylmethyl-rhodobacteriochlorin    13¹,17³-di(2-trimethylammoniumethyl)amide (compound 23).

The above compounds may be prepared as described in WO 2005/120573.

In certain embodiments, the positively charged photosensitizer used inaccordance with the invention is a Bchl derivative of the formula Iwherein M is Pd, R₂ is —COOCH₃, R₃ is H, R₄ is —COCH₃, R₅ is ═O, and R₁is —OR₈, wherein R₈ is a residue of an amino acid containing an hydroxygroup, preferably serine, or a derivative thereof, preferably an alkyl,more preferably methyl, ester, or a peptide containing said amino acidor derivative thereof, in which amino acid residue the free amino groupmay be quaternized as a trimethylammonium group. An example of suchderivative of formula I is the herein designated compound 13:O—[Pd-Bpheid]-[N³-trimethylammonium-2-methyl]-Serine methyl ester iodidesalt.

In accordance with the present invention, pharmaceutically acceptablesalts of the (bacterio)chlorophyll compounds of formula I-III are usedfor PDT treatment of the eye, both salts formed by any carboxy groupspresent in the molecule, and a base as well as acid addition salts.

Pharmaceutically acceptable salts are formed with metals or aminescations, such as cations of alkali and alkaline earth metals or organicamines. Examples of metals used as cations are sodium, potassium,magnesium, calcium, and the like. Examples of suitable amines areN,N′-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine,ethylenediamine, N-methylglucamine, and procaine (see, for example,Berge S. M., et al., “Pharmaceutical Salts,” (1977) J. of PharmaceuticalScience, 66:1-19).

Acid addition salts of basic photosensitizers include salts derived frominorganic acids such as hydrochloric, nitric, phosphoric, sulfuric,hydrobromic, hydriodic, phosphorous, and the like, as well as saltsderived from organic acids such as aliphatic mono- and dicarboxylicacids, phenyl-substituted alkanoic acids, hydroxy alkanoic acids,alkanedioic acids, aromatic acids, aliphatic and aromatic sulfonicacids, etc. Such salts thus include sulfate, pyrosulfate, bisulfate,sulfite, bisulfite, nitrate, phosphate, monohydrogenphosphate,dihydrogenphosphate, metaphosphate, pyrophosphate, chloride, bromide,iodide, acetate, propionate, caprylate, isobutyrate, oxalate, malonate,succinate, suberate, sebacate, fumarate, maleate, mandelate, benzoate,chlorobenzoate, methylbenzoate, dinitrobenzoate, phthalate,benzenesulfonate, toluenesulfonate, phenylacetate, citrate, lactate,maleate, tartrate, methanesulfonate, and the like. Also contemplated aresalts of amino acids such as arginate and the like and gluconate orgalacturonate (see, for example, Berge S. M., et al., “PharmaceuticalSalts,” (1977) J. of Pharmaceutical Science, 66:1-19).

The acid addition salts of said basic compounds are prepared bycontacting the free base form with a sufficient amount of the desiredacid to produce the salt in the conventional manner. The free base formmay be regenerated by contacting the salt form with a base and isolatingthe free base in the conventional manner. The free base forms differfrom their respective salt forms somewhat in certain physical propertiessuch as solubility in polar solvents, but otherwise the salts areequivalent to their respective free base for purposes of the presentinvention.

The base addition salts of acidic photosensitizers used in accordancewith the invention are prepared by contacting the free acid form with asufficient amount of the desired base to produce the salt in theconventional manner. The free acid form may be regenerated by contactingthe salt form with an acid and isolating the free acid in theconventional manner. The free acid forms differ from their respectivesalt forms somewhat in certain physical properties such as solubility inpolar solvents, but otherwise the salts are equivalent to theirrespective free acid for purposes of the present invention.

The negatively charged and the positively charged chlorophyll andbacteriochlorophyll compounds and pharmaceutically acceptable saltsthereof used in accordance with the invention are highly water solubleand enable the preparation of aqueous formulations with no addedsurfactants.

The pharmaceutical composition used in accordance with the invention isan ophthalmic composition, preferably a liquid e.g., a solution,suspension, and emulsion, or a gel that is topically applied to the eyeof the patient and allowed to penetrate the cornea or the sclera beforePDT is applied. The ophthalmic composition usually comprises inertpharmaceutically acceptable carriers and excipients that are well knownin the art. The pharmaceutical composition may further contain one ormore functional excipients, for example, for the purpose of mediatingthe depth of active agent penetration. Limiting the depth ofphotosensitizer penetration may be advantageous as it confines thetreatment to the region of interest thereby minimizing adverse effectsto adjacent tissues caused by the reactive oxygen species (ROS) formedupon illumination of the photosensitizer.

For preparation of pharmaceutical compositions, the photosensitizers maybe lyophilized, for example, with mannitol, and the dry powder isdirectly solubilized in saline or any other pharmaceutically acceptableaqueous solution for topical application to a patient or for applicationon a sample in vitro target. The preparation of the compositions iscarried out by techniques well-known in the art, for example assummarized in Remington: The Science and Practice of Pharmacy, MackPublishing Co., Easton, Pa., 1990.

The present inventors found that penetration of severalbacteriochlorophyll derivatives into rabbit corneas and sclera that wereexposed to the photosensitizer for 10 or 30 min was significantly deeperwhen the photosensitizer was applied without dextran-500 than whenapplied with the excipient. Fluorescence microscope measurements ofrabbit cornea presented in FIGS. 4A-4H indicate that the negativelycharged bacteriochlorin derivative WST11 crosses half of the cornealstroma after 10 minutes of incubation and the entire stroma depth after30 min of incubation in the absence of dextran. In contrast, applicationof WST11 with dextran-500 limited the penetration depth to the outer ⅓of the corneal stroma after 10 minutes and to ˜50% after 30 minutes ofincubation. Similar results were obtained when the penetration depth ofother sulfonated Bchls derivatives, namely compounds 1-7, was measuredin the presence of dextran in the formulation.

Treatment with WST11 (or with compounds 1-7) mixed with dextran-500 didnot alter the stiffening effect as compared to WST11 without dextran.However, it greatly reduced the treatment's adverse effects. Clinically,the duration and extent of corneal edema and epithelial healing wassignificantly shortened. Also, epithelial haze that formed in sometreated corneas, appeared only in the absence of dextran. However, themost important effect appears to be the protection of the endotheliallayer and the reduced impairment of keratocytes in the treated corneas.This is clearly demonstrated in the histological sections of rabbit eyespresented in FIGS. 11A-11C herein. Histology tests after treatment withWST11 (or with compounds 1-7) admixed with dextran 500, followed by NIRillumination showed reduction in keratocyte population in the anteriorcornea with no damage to the endothelium.

This protection as well as the attenuated edema and shortened time ofhealing are probably related to the reduced depth of WST11 penetrationand subsequently, the spatially limited effect of the photogeneratedradicals. Surprisingly, while WST11 in saline penetrated half of thecorneal stroma after 10 minutes of incubation, it produced lessstiffening than WST11 applied with dextran after penetrating to asimilar depth in the cornea. This observation may qualitativelycorrelate with the accumulation of WST11 at the cornea central layer andthe possibility to generate a higher ROS concentration in this domaincompared to the more diffused distribution of WST11 in the absence ofdextran.

The protective role of dextran is probably maintained in the riboflavin(RF)/UVA treatment known in the art for stiffening cornea. Althoughformulation of RF with dextran-500 has been claimed to help maintainingsolution isoosmolarity (Letko et al., 2011), it was recently reportedthat the stromal penetration of RF-dextran solution (RF-D) is limited tothe anterior 200 μm, even under prolonged exposure time (Søndergaard etal., 2010). This finding corroborates with data obtained by the presentinventors and substantiates the conclusion that dextran limits theanterior penetration of the photosensitizer into the cornea and therebyprotects the endothelium from phototoxic effect. The molecular nature ofthis effect is yet to be determined but it emerges that thepolysaccharide forms together with the natural colagen a matrix, whichacts as a barrier that prevents migration of the photosensitizer intodeeper layers.

Dextran is a high molecular weight polymer of glucose, digestible anddegradable, which accounts for its wide use as an excipient forophthalmic formulations, for example, artificial tears and eye drops.Dextran fractions are derived from the partial acid hydrolysis of nativedextran, they have various properties, and uses, and are supplied inmolecular weights ranging from 1000 to 2 million. The molecular weightof the fraction is in most cases a key property. The designation dextran5, dextran 10 and the like represents the mean molecular weight dividedby 1000. Thus, Dextran 10 corresponds to a mean molecular weight of10,000. In certain embodiments, the dextran fractions are selected fromdextran 50, dextran 70, dextran 100, dextran 200, dextran 500, dextran1000 and dextran 2000. The most preferable dextran fraction used for thepreparation of the ophthalmic composition of the invention is dextran500.

When a 20% dextran 500 is added to the formulation it increases theviscosity of the formulation to about 179 cP. An alternative approach toexplain the reduced penetration depth of the photosensitizer whenco-administered with dextran is increased viscosity of the formulation.The present inventors tested the effect of co-administering thephotosensitizer with a solution of 65% glucose or 92% glycerol having aviscosity of 200 cP, comparable to that of 20% dextran solution. Itturned out that a solution of 65% glucose or 92% glycerol limited thepenetration depth of the photosensitizer to the same extent as dextran.

Thus, in more preferred embodiments, the present invention provides aviscous ophthalmic composition for treatment of corneal thinning.According to these embodiments, the ophthalmic composition comprises aviscous agent as an excipient that enhances the viscosity of theformulation and thereby restricts or delays the active agent penetrationinto the cornea. Any viscous agent used in ophthalmic formulations issuitable for the purpose of the invention. Preferred functionalexcipients that function as viscous agents are biopolymers, preferablypolysaccharides, more preferably natural polysaccharides that are highlybiodegradable in the body as a result of natural biological processes.Natural polysaccharides are advantageous also due to their uniquephysico-chemical properties, relatively low cost and since they can bechemically modified to suit specific needs. Non-limiting examples ofpolymeric viscous agents include dextran, scleroglucan and derivativesthereof, Gellan gum, Guar gum, methylcellulose (MC), polyvinyl alcohol,polyvinyl pyrrolidone, propylene glycol, polyethylene glycol, gelatin,carbomers and the like. Preferred viscous agents are dextran, and theexopolysaccharide hydrogels scleroglucan, particularly carboxylatedderivatives thereof, Gellan gum and Guar gum.

Non-polymeric viscous agents that may be used in the ophthalmicformulation of the invention include glucose and glycerol.

Alternatively, the composition may be co-administered with a viscous,inert solution, such as, but not limited to, a solution of 20% dextranor 65% glucose or 92% glycerol.

The optical application of the bacteriochlorophyll derivatives in rabbitcornea and sclera followed by illumination at 600-900 nm, and thetrapping of photogenerated oxygen radicals in the cornea and sclera,induced photochemical reactions, probably polymerization reactions, thatresulted in consistent corneal and scleral stiffening ex vivo and invivo. The photochemical reactions of the bacteriochlorophyll derivativesapplied to the eyes of rabbits could be observed as a continuousbleaching and spectral modifications during illumination (the bleachingis shown herein in FIG. 5).

For rabbit cornea, ex vivo incubation with WST11 for 30 minutes prior toillumination increased the Young's modulus and the ultimate stress by369% and 267%, respectively. Treatment with the same parametersperformed in vivo raised the Young's modulus and ultimate stress by 174%and 111%, respectively. The Young's modulus increased with theincubation time for the cornea treated in vivo.

For rabbit sclera, ex vivo incubation with WST11 for 30 minutes prior toillumination, increased the Young's modulus and the ultimate stress by300% and 274%, respectively.

Thus, PDT of cornea and sclera pre-treated with water soluble(bacterio)chlorophyll derivatives enhanced the ultimate stress and theYoung's modulus by at least a factor of 2, with no damage to endothelialcells. This treatment appeared safe in the animal models and maytherefore be considered for clinical trials in keratoconus and cornealectasia after refractive surgery.

Thus, in another aspect, the present invention provides a method forphotodynamic therapy of eye diseases, disorders and conditionsassociated with corneal thinning or scleral stretching, comprising thesteps of: (a) administering to an individual afflicted with cornealthinning or sclera stretching a photosensitizer which is a(bacterio)chlorophyll of the formula I, II or III as defined above or apharmaceutical composition comprising same; and (b) irradiating the eyewith light at the red or near infrared (NIR) wavelength.

The method provided by the invention is advantageous over RF/UVAtreatment. UVA irradiation was shown by several reports to be toxic tocorneal endothelial cells (Wollensak et al., 2004(a); Wollensak et al.,2004(b); Wollensak 2010(a)) and to result in a complete loss ofkeratocytes (Spoerl et al, 2007; Hafezi et al., 2009; Wollensak et al.,2010(b)) even when the penetration depth of the applied RF was limited.

The mechanism involved in corneal stiffening by bacteriochlorophyll/NIRtreatment may differ from that imposed by RF/UVA. Photoexcited watersoluble bacteriochlorophyll derivatives such as WST11 generatesuperoxide and hydroxyl radicals with minimal traces of singlet oxygenwhich is the major ROS product of RF/UVA treatment (see FIG. 6).

In one embodiment, the method of the invention is applied for treatmentof corneal thinning diseases, disorders and conditions selected fromkeratoconus, corneal ectasia caused by trauma, for example,post-laser-assisted in situ keratomileusis (LASIK) ectasia,post-photorefractive keratectomy (PRK) ectasia, post-infection ectasia,peripheral ectasia, atrophy, raised intraocular pressure or as acomplication of photorefractive surgery in which the corneal stroma hasbeen left thinner than about 250 μm.

Other non-limiting diseases and conditions associated with visual lossas a result of scleral and/or cornea anomalies that can be treated bythe methods of the invention include rheumatoid condition of the cornea,degenerative myopia, regular myopia, scleral staphyloma, ocularhypertension glaucoma, low tension glaucoma and combinations thereof.

The combined treatment of (bacterio)chlorophyll photosensitizer and redor NIR irradiation of the eye may be used for preventing expectedscleral weakening or corneal thinning in patients that intend to, oralready underwent interventional procedures.

Thus, in a further aspect, the invention provides a method forpreventing a corneal and/or scleral disease or weakening before, duringor after interventional procedures, comprising the steps of: (a)administering to an individual expected to be afflicted with cornealand/or scleral disease or weakening, a chlorophyll orbacteriochlorophyll compound as defined above or a pharmaceuticalcomposition comprising same; and (b) irradiating the eye with light at ared or near infrared (NIR) wavelength.

The amount of (bacterio)chlorophyll derivative to be administered forPDT therapy will be established by the skilled physician according tothe experience accumulated with other Bchl derivatives used in PDT, andwill vary depending on the choice of the derivative used as activeingredient, the condition to be treated, the mode of administration, theage and condition of the patient, and the judgement of the physician.

EXAMPLES Materials and Methods

(i) Compounds.

Palladium 3¹-oxo-15-methoxycarbonylmethyl-rhodobacteriochlorin13¹-(2-sulfoethyl)amide monovalent salt (e.g. the dipotassium saltWST11) was supplied by STEBA Laboratories, Israel. Palladium3¹-oxo-15-methoxycarbonylmethyl-rhodobacteriochlorin13¹-(-sulfopropyl)amide divalent salt (e.g., the dipotassium saltcompound 1); palladium3¹-oxo-15-methoxycarbonylmethyl-rhodobacteriochlorin13¹,17³-di-(2-sulfoethyl)amide divalent salt (e.g., the dipotassium saltcompound 2); palladium3¹-oxo-15-methoxycarbonylmethyl-rhodobacteriochlorin13¹,17³-di-(3-sulfopropyl)amide salt (e.g., the dipotassium saltcompound 3); 3¹-oxo-15-methoxycarbonylmethyl-rhodobacteriochlorin13¹-(2-sulfoethyl)amide salt (e.g., the dipotassium salt compound 4);3¹-oxo-15-methoxycarbonylmethyl-rhodobacteriochlorin13¹-(-sulfopropyl)amide salt (e.g., the dipotassium salt compound 5);3¹-oxo-15-methoxycarbonylmethyl-rhodobacteriochlorin13¹,17³-di-(2-sulfoethyl)amide divalent salt (e.g., the dipotassium saltcompound 6) and 3¹-oxo-15-methoxycarbonylmethyl-rhodobacteriochlorin13¹,17³-di-(3-sulfopropyl)amide divalent salt (e.g., the dipotassiumsalt compound 7), were prepared as described in U.S. Pat. No. 7,947,672.Other bacteriochlorophyll derivatives e.g., derivatives containingnegatively charged groups or positively charged groups were prepared asdescribed in U.S. Pat. No. 7,947,672 and WO 2005/120573. Solutions ofthe bacteriochlorophyll derivatives were prepared in two forms: (a) insaline only, at a concentration of 2.5 mg/ml and pH adjusted to 7.3(herein referred to as “Bchl-S solution” or “Bchl-S”); (b) 2.5 mg/ml ofa bacteriochlorophyll derivative in saline with 20% dextran 500 (31392Fluka, Switzerland) and pH adjusted to 7.3 (herein referred to as“Bchl-D solution” or “Bchl-D”).

Two forms of riboflavin (RF) solution were used: (a) 0.1% solution ofriboflavin-5′-phosphate in saline, pH adjusted to 7.3 (herein referredto as “RF solution”); (b) commercial (Medio Cross, Germany) 0.1%solution of riboflavin-5′-phosphate in 20% dextran T-500, measured pH6.8 (herein “RF-D solution” or RF-D″).

(ii) Light Sources.

(a) Diode laser with tunable output up to 1 W at 755 nm (CeramOptec,Germany); (b) LED system at 760 nm 2×18 mW (Roithner Lasertechnik,Austria); and (c) LED system at 370 nm 2×3 mW (Roithner Lasertechnik,Austria).

(iii) Animal.

New Zealand white (NZW) rabbits were housed and handled with ad libitumaccess to food and water at the Core Animal Facility of the WeizmannInstitute of Science (Rehovot, Israel). All experimental procedures wereapproved by the Institutional Animal Care and Use Committee. Allexperiments were done in adherence to the ARVO Statement for the Use ofAnimals in Ophthalmic and Vision Research.

Methods

(a) Corneal Studies

Biomechanical Testing of Corneal Stiffness

(i) Sample Preparation for Ex Vivo Biomechanical Studies of CornealStiffening.

Eyes of rabbits (15-16 weeks old, weighting 3-4 Kg) were enucleated postmortem. Before enucleation, the 12 and 6 h position were marked on thesclera for preservation of subsequent cutting orientation. The corneaswere de-epithelialized mechanically using a PKR scraper (BectonDickenson, USA). Usually, about 10 rabbits were assigned tobacteriochlorophyll/NIR treatment, herein the “test group” (for exampleWST11/NIR), and 2 rabbits were treated by RF/UVA (herein the “RF/UVAgroup”) for comparison. In the test group, 10 eyes of 10 rabbits wereimmersed upside down for 30 minutes in test compound saline solution(Bchl-S) followed by NIR illumination (600-900 nm) with a diode laser at10 mW/cm² for 30 minutes. The untreated contralateral eyes served ascontrols. In the RF/UVA group, RF-D solution was topically applied on 2eyes of 2 rabbits, every 10 minutes, for 30 minutes, followed by UVAirradiation at 370 nm by a double diode at 3 mW/cm², for 30 minutes.After treatment, the corneo-scleral rings were removed and placed onparaffin hemisphere buttons with a matching shape to establish accuratesectioning without tissue streching. Corneal strips, 4±0.2 mm in width,were cut from the epithelial side in a superior-inferior orientation,with a self-constructed double-blade cutter. The strips included 2 mm ofsclera on both ends. Central corneal thickness was measured byultrasonic pachymetry (Humphrey ultrasonic pachymeter, USA). The cornealstrips were then transferred to the biomechanical tester.

(ii) Sample Preparation for In Vivo Studies

Twelve to twenty five weeks old rabbits (2.5-3 Kg weight) were used.They were anesthetized by intramuscular (i.m.) injection of 35 mg/kgketamine (Rhone Merieux, Lyon, France) and 5 mg/kg xylazine (Vitamed,Binyamina, Israel). Usually, 16 rabbits were assigned forbacteiochlorophyll treatment. After de-epithelialization, one eye ofeach rabbit was treated topically with a test compound in saline for 10,20 and 30 minutes using an eye cap (12 mm in diameter), followed by NIRillumination for 10, 20 or 30 minutes (755 nm, 10 mW/cm²). The other eyeserved as untreated control. To prevent exposure of the limbal stemcells, the illuminated area was restricted by an aluminum foil mask withan 8 mm diameter central opening. To determine the role of dextran, 12rabbits were divided into 4 treatment groups of 3 rabbits each: group 1,the Bchl-S group, was treated with a saline solution of abacteriochlorophyll derivative; group 2, the Bchl-D group, treated witha bacteriochlorophyll-dextran solution. Both groups were incubated withthe test compound for 20-min, followed by NIR illumination (755 nm, 10mW/cm²) for 30 minutes. Group 3, the RF group, was treated withriboflavin without dextran, and group 4, the RF-D group was treated withRF-dextran solution (RF-D). Both groups were incubated with RF or RF-Dsolutions for 30-minute, followed by UVA illumination (370 nm, 3 mW/cm²)for 30 minutes. An ophthalmic ointment containing dexamethasone 0.1%,neomycin and polymixin B (Maxitrol®, Alcon, Belgium) was applied on thetreated eyes once daily for two weeks. Four weeks after the treatmentthe rabbits were sacrificed, and the corneoscleral rings were removedand placed on paraffin hemisphere buttons of matching shape to assureaccurate sectioning without stretching the tissue. Corneal strips, 4±0.2mm in width, were cut from the epithelial side, as described above inthe ex-vivo section. The corneal strips were transferred to thebiomechanical tester without delay.

Drug Accumulation, Penetration and Photobleaching

(i) Photosensitizer Overall Accumulation

Eyes of rabbits were de-epithelialised mechanically immediately postmortem. For most studies, 6 rabbits were used, wherein 6 eyes of 6rabbits were exposed to the bacteriochlorophyll test compound for 10, 20and 30 minutes (2 eyes for each test) using an eye cap, while thecontralateral eyes were left untreated and served as controls. Thecorneas were removed and the central buttons of 8-mm diameter werepunched out with a round trephine and placed onto the outer side of apolymethylmethacrylate cuvette in the area of light beam passage.Absorption spectra were recorded, and optical density (OD) at 600-900 nm(in most cases at 755 nm) was measured using V-570 spectrophotometer(Jasco, Japan).

(ii) Depth Penetration Studies

Eyes of rabbits were enucleated and deepithelialised mechanically postmortem. The eyes were exposed to Bchl-S solution using an eye cap for 5,10, and 30 minutes and to Bchl-D solution for 10 and 30 min, indarkness. Controls: untreated eyes, and one eye treated with dextran-500only for 30 min in darkness. Following this pretreatment, the corneaswere briefly rinsed with saline and the central 8-mm buttons weretrephined, removed, wrapped in aluminum foil and frozen on dry ice untilfurther use. Central serial corneal sagittal slices (12 μm) were cutwith a cryomicrotome, mounted on a microscope glass slide and storedfrozen in −70° C. until use. For measurement of fluorescence, theindividual slices were placed on the microscope base and a photographicrecord was immediately taken. Fluorescence intensity of Bchl-S or Bchl-Dtreated corneas from 3 serial cryo sections was recorded at 760 nm, uponexcitation at 740 nm, using a fluorescence microscope (BX61 Olympus,Japan) equipped with CCD camera (Cascade 512B, Roper Sci., USA) and along pass filter of above 760 nm. The digital data was analyzed, usingImageJ software (NIH, USA).

(iii) Photobleaching of Photosensitizes

Corneas of eyes from euthanized rabbits were de-epithelialized. Usually,5 rabbits were tested, and 8 of the 10 eyes were pretreated for 20minutes with a bacteriochlorophyll derivative in saline (Bchl-S), usingan eye cap. The solution excess was carefully tipped off with filterpaper. The eyes were irradiated for the specified time duration: 0, 10,30, and 60 min (two corneas per time frame). Additional two eyes wereused as control. The corneas were removed, and the central buttons of8-mm diameter were punched out with a round trephine. Absorption spectrawere recorded using V-570 spectrophotometer (Jasco, Japan).

Fluorescence Spectroscopy Following Bchl/NIR and RF/UVA Treatment

Rabbit eyes were enucleated post mortem. The corneal epithelium wasde-epithelialised with a scraper. Usually, two eyes of two rabbits wereimmersed in Bchl-S or Bchl-D solutions for 30 min, followed by NIRirradiation (30 min). RF-D solution was applied on two eyes for 30 min,followed by UVA irradiation (30 min). Two eyes served as controls. Thecorneas were removed and the central 8-mm buttons were punched out witha round trephine. The buttons were mounted on a glass slide that wasplaced obliquely at 45° (FIG. 2) in a spectrofluorimeter (Varian-CaryEclipse, USA). Excitation beam was set at 295, 315, 320, 328, 335 nm andcorresponding emission spectra were read at 360 to 480 nm. Otherreadings were performed with excitation at 350 nm and emission at380-480 nm, and excitation at 370 nm and emission at 400-700 nm.Excitation slit was 10 nm, emission slit ˜10 nm.

Histology

Forty eight hours or 1 week after treatment with Bchl-S, Bchl-D, RF orRF-D, rabbits were euthanized, and the eyes were immediately enucleatedand fixated in Davidson's fixative (48 hour post treatment) or in 4%formaldehyde (1 week post treatment). Six-μm sections were prepared fromeither the whole eye or corneas and stained with hematoxylin-eosin(H&E). The pathology of the corneal and retinal sections was examined.The sections were photographed with a digital video camera (NikonDS-Ri1, Japan), mounted on a light microscope (E-800 Leica, Germany).Keratocyte and endothelial cell densities were counted in 2 areas of 2central histological sections and calculated, using Image-Pro software(MediaCybernetics, MD, USA).

Staining for Apoptosis.

For apoptosis detection, rabbits were euthanized one day post Bchl-S orBchl-D treatment. Their corneas were removed, fixated in 4% formaldehydeand embedded in paraffin. Six-μm central sections were prepared from thecorneas, placed on microscope slides and deparaffinized.Peroxidase-based terminal deoxyribonucleotidyl transferase-mediateddUTP-digoxigenin nick and labeling (TUNEL) assay was performed accordingto the manufacturer's instructions (Apop-Tag assay, Chemicon, MerckMillipore, Darmstadt, Germany).

Endothelial Staining.

Rabbits underwent analysis of corneal endothelial damage 1 day posttreatment, using alizarine red S and trypan blue staining as describedin Spence and Peyman, 1976. The treatment included either a 20-minutepretreatment with Bchl-S or Bchl-D solutions, followed by 30-minuteirradiation at 755 nm (2 rabbits), or only a 30-minute irradiation at760 nm (2 rabbits). Contralateral non-treated eyes served as controls.

(b) Scleral Studies

Sample Preparation for Ex Vivo Biomechanical Studies of ScleralStiffening

Rabbit eyes were enucleated immediately post mortem and treatedexternally by applying an eye cap with a 2.5 mg/ml of a test compound insaline (Bchl-S) for 30 minutes on the superior or inferior sclera,followed by irradiation with NIR at 10 mW/cm² for 30 minutes. Theopposite side of the sclera served as control. Scleral strips, 4 mm inwidth, were cut at the equatorial treated area and the opposite sclera,and stress-strain measurements were performed using a biomaterialtester.

Impregnation Measurements

Ex-Vivo Impregnation:

Eyes of euthanized rabbits were enucleated, and the superior equatorialsclera (or inferior equatorial) was exposed to a test compound (Bchl-S)solution (2.5 mg/ml) for 10 minutes and 30 minutes using an eye cap of10 mm diameter filled with the test compound. The inferior equatorialsclera (or the superior equatorial sclera, if impregnation was appliedto the inferior equator) served as control.

In Situ Impregnation:

Test compound solution was applied in the supero-temporal orinfero-nasal (alternating) quadrant of the rabbit by a curved plastic ormetal glide with attached Merocel® sponge connected by a tube that ranalong the glide outside. The glide was inserted through a conjunctivalopening at the limbus (see FIGS. 17A-17B). After placing the glideattached to the sclera, the tube was connected to a syringe or pump, andthe test compound reservoir was injected to impregnate the sclera.Application of the photosensitizer was continued for 10 or 30 minutes.Afterwards, the glide with the Merocel® was withdrawn.

The following technique was performed after ex-vivo or in-situimpregnation of photosensitizer:

The treated scleral area and the opposing control sclera were trephinedwith a round skin trephine of 8-mm diameter and scissors and immediatelyfrozen. Sagittal slices of 20 microns were dissected with acryomicrotome at the central area of the scleral button. Thefluorescence intensity at 755 nm was recorded upon excitation at 740 nmusing a fluorescence microscope (Olympus, Japan).

Illumination In Vivo Through the Anterior Segment.

NIR illumination was performed through the cornea and lens by a diodelaser with a fundus contact lens (Volk pan-fundoscopic) or by mountingthe optic fiber to an indirect ophthalmoscope, and using a +20 diopterlens applying the light on the sclera through the retina and pigmentepithelium. The illumination intensity was adjusted accordingly due tothe attenuation of the light.

After illumination, the rabbits were euthanized, their eyes wereenucleated, and the treated scleral area and the opposing control sclerawere trephined with a round skin trephine of 8-mm diameter and scissors.The scleral buttons were frozen immediately. Sagittal slices of 50microns were performed with a cryomicrotome at the central area of thescleral button. Fluorescence spectroscopy was performed using afluorescence microscope (Olympus, Japan), and fluorescence intensity at755 nm was recorded upon excitation at 740 nm.

(c) Biomechanical Testing

The corneal and scleral strips were clamped horizontally, at a distanceof 6 mm, between the jaws of a microcomputer-controlled biomaterialtester (Minimat, Germany) with a 200 Newton force cell (FIG. 1).Controlled tightening of screws was performed with a calibratedscrewdriver at a preset torque of 9 cN·m (Torqueleader, England). Thestrain was increased linearly at a rate of 1.0 mm/min and was measuredup to tissue rupture. Young's modulus was calculated by the testingmachine. Ultimate stress and Young's modulus were expressed in MegaPascal (MPa) units.

(d) Electron Spin Resonance (ESR) Spectroscopy

All ESR measurements were carried out using a Magnettech ESR MiniscopeMS 100 Spectrometer (Germany), with a Microwave X-band Bridge. The ESRspectrometer operates at 9.3-9.55 GHz, 20 mW microwave power. ESRmeasurements were carried out at room temperature in glass capillariesor flat cells.

Samples of aqueous solutions of bacteriochlorophyll derivatives withoutor with dextran (Bchl-S and Bchl-D, respectively), and of riboflavinwithout or with dextran (RF and RF-D, respectively), were illuminated at755 nm as described in Ashur et al, 2009. To each solution, thespin-trap α-(4-pyridyl N-oxide)-N-tert-butylnitrone (4-POBN, 65 mM) andethanol (8%) were added. Controls contained illuminated 4-POBN in salinewith/without dextran, and non-illuminated Bchl-S, Bchl-D, RF and RF-Dsolutions with/without 4-POBN.

Ex-Vivo ESR Measurements of Rabbit Corneas.

Eyes of rabbits were enucleated post mortem. The corneas werede-epithelialized mechanically and corneal strips, 5 mm in width, werecut with a self-constructed double-blade cutter. The strips wereimmediately immersed for 30 mM in solutions containing 4-POBN (65 mM)and ethanol (8%), and either Bchl-S or RF-D. Next, the strips werewashed with saline and put in the flat-cells (0.5×5 mm³) for NIR/UVAillumination followed by ESR measurements with the aforementionedMiniscope.

Example 1 WST11 Uptake by Rabbit Corneal Tissue

(i) Overall Accumulation of WST11 by the Rabbit Cornea

Six eyes of 3 euthanized rabbits were de-epithelialized mechanicallyimmediately post mortem. Five of these eyes were exposed to WST11 insaline (WST11-S solution) for 10, 20 and 30 min, using an eye cap, andthe optical absorption at 755 nm of the washed corneas was determined asdescribed in Materials and Methods. One eye served as untreated control.

As shown in FIG. 3, the optical density of the impregnated corneasincreased with time up to a value of 1.71 OD units, without interferencewith the native spectrum of WST11.

(ii) WST11 Depth of Penetration into the Rabbit Cornea

Upon topical application, the penetration and the depth of photochemicalimpact on the corneal tissue was determined for severalbacteriochlorophyll derivatives. Results are provided herein formeasurements with WST11.

The penetration depth of the photosensitizer determines the extent ofcornea's tissue exposure to the photodynamic effect. In particular, thedeeper the penetration, the higher is the probability for endothelialimpairment. To resolve the penetration depth of test compounds followingdifferent times of incubation, the digitized fluorescence of dissectedcorneal discs was monitored using fluorescence microscopy as describedin Materials and Methods.

The penetration depth of WST11 into the de-epithelialized cornea,following various incubation times, was performed in the darkness in theabsence or presence of dextran, using saline or dextran solutions ofWST11 (WST11-S or WST11-D solutions, respectively) in 20 eyes ofeuthanized rabbits. Sagittal frozen sections mounted on glass slideswere subjected to fluorescence microscopy. The distribution of WST11across the cornea was recorded photographically by fluorescencemicroscopy (excitation/emission 740/760 nm). The controls were 4untreated eyes and 1 eye treated only with dextran-T 500 for 30 minutes.

Following 10 minutes of exposure (of 2 eyes) to WST11-S solution, WST11was evident through the entire outer half of the stroma (FIGS. 4A, 4C).Further exposure (30 minutes; 5 eyes) resulted in diffused fluorescenceall the way to Descemet's membrane (FIGS. 4B, 4D). This treatmentincreased the stromal thickness from 360-380 μm to 600-730 μm. Incontrast, application of WST11-D solution limited the penetration depthto the outer ⅓ (200 out of 520 μm) of the corneal stroma, at both 10 and30 minutes exposure times (3 eyes and 5 eyes were tested, respectively)as shown in FIGS. 4E, 4G and FIGS. 4F, 4H, respectively. Importantly,the fluorescence of WST11-D in the cornea showed a relatively sharpfront of drug migration that did not exceed the center of the stroma.However, at the depth of 200 μm the level of fluorescence was stillhigher for corneas following 30 minute exposure to WST11-D suggesting ahigher accumulation level.

Comparable results for corneal impregnation and penetration depth wereobtained with saline and dextran solutions of compounds 1-7. (data notshown).

Example 2 Photochemistry of WST11 in the Cornea

(i) Photobleaching of WST11 in Ex-Vivo Treated Corneas

It was previously described by the present inventors that generation ofoxygen radicals upon illumination of certain bacteriochlorophyllderivatives such as WST11 in the absence of serum albumin is accompaniedby bleaching of the NIR transition and absorption increase at ˜645 nmdue to the photochemical generation of a chlorophyll-like molecules(Ashur et al. 2009). Such changes are therefore important markers forthe photochemical activity of such bacteriochlorophyll derivatives insitu. Hence, the changes in the optical absorption of several sulfonatedbacteriochlorophyll derivatives during cornea illumination weremonitored in de-epithelialized corneas of rabbits. Results obtained for10 corneas of 5 euthanized rabbits treated with WST11 in saline arepresented herein.

Eight de-epithelialized eyes were retreated with WST11-S for 20 minusing an eye cap, and the remaining 2 eyes served as control. As shownin FIG. 5, the 755-nm absorption of corneal WST11 diminished by 30, 50and 75% after NIR illumination for 10, 30 and 60 min, respectively(downward arrow). In parallel, the absorption increased at 645 nm(upward arrow), typical to the corresponding chlorin derivativeformation by the photochemical interaction of WST11 with molecularoxygen (Ashur et al., 2009)

(ii) Oxygen Radical Formation by Photoexcited WST11 in the CorneaDeduced from Electron Spin Resonance (ESR) Spectroscopy

Comparison of the ESR spectra of α-(4-pyridylN-oxide)-N-tert-butylnitrone (4-POBN) in aqueous solution followingphotoproduction of reactive oxygen species (ROS) by WST11/NIR (black)and RF/UVA (light blue) and spin trapping by 4-POBN, are presented inFIG. 6. The observed quartet due to singlet oxygen trapping is presentonly in the RF/UVA spectra, while the sextet represents superoxide andhydroxyl radical formation (stars) in both WST11/NIR and RF/UVA.

ESR measurements of ROS trapped in the rabbit cornea treated byWST11/NIR is shown in FIG. 7. The signals are similar to those observedin aqueous solutions with no traces of singlet oxygen. The signalsgenerated by RF/UVA were at the noise level.

Example 3 Corneal Stiffening in Response to WST11-S/NIR Treatment

Stress-Strain Measurements of Ex-Vivo Treated Eyes

Stress-strain measurements were conducted in 12 rabbits as described inMaterials and Methods. Briefly, 10 eyes of 10 rabbits were treated byWST11/NIR, and 2 eyes of 2 rabbits were treated by RF-D/UVA. Thecontralateral eyes served as control.

The stress-strain measurements following 30 min pre-incubation withWST11-S and then NIR illumination, showed a nearly 3-fold increase inthe corneal stiffness (of all 10 eyes) as compared to the untreatedcontrol eyes (FIGS. 8A-8B). There was a maximal increase of 267% in theultimate stress (P<0.0001) from a mean of 1.63 MegaPascal (MPa) withouttreatment to 5.98 MPa after treatment and an increase of 369% in Young'smodulus (P<0.0001), from a mean of 3.76 MPa without treatment to 17.65MPa after treatment. The results are depicted in Table 1.

The mean ultimate stress in the two RF-D/UVA-treated corneas increasedfrom 1.44 MPa without treatment to 6.46 MPa after treatment and in themean Young's modulus from 3.28 MPa without treatment to 20.72 MPa aftertreatment.

Thus, stiffening due to WST11/NIR treatment appeared similar to thatobserved in the two RF-D/UVA treated corneas.

Stress-Strain Measurements of In-Vivo Treated Eyes

Corneas of 16 live rabbits were pretreated for 10 (n=4), 20 (n=6) and 30(n=6) minutes with WST11-S (2.5 mg/ml). NIR illumination (755 nm, 10mW/cm²) was then delivered for 30 minutes. The eyes were allowed toheal, and one month later the ultimate stress of the treated corneas wasmeasured (as described in Material and Methods), and found to increaseby 45, 113, and 126%, respectively, as compared to the non-treated eyes.The mean Young's modulus in WST11-S/NIR-treated corneas showed a 10, 79and 173% increase for the same treatment times. Results are shown inFIGS. 9A-9B, and in Tables 1 and 2.

Corneas treated by WST11-S/NIR developed edema for one week aftertreatment. The corneal epithelial defect healed gradually after 10-14days. After epithelial healing, the corneas regained transparency, withsome corneas demonstrating epithelial haze.

TABLE 1 rabbit cornea stiffening following WST11-S/NIR treatment Meanultimate stress Mean Young's Modulus Intensity Increase % IntensityIncrease % Setting Sample (MPa) (P value) (MPa) (P Value) Ex Control:untreated fellow 1.63 ± 0.58  3.76 ± 1.96 vivo eyes n = 10 WST11/NIRtreated eyes 5.98 ± 2.11 286 17.65 ± 8.25 410 (<0.0001) n = 10 (<0.0001)In vivo Control: untreated fellow 3.06 ± 0.67 11.7 ± 2.6 30 eyes n = 6days WST11/NIR treated eyes 6.91 ± 0.53 111   32 ± 3.6 174 (0.0035)  n =6 (0.0049)

TABLE 2 Effect of WST11 incubation time on rabbit corneas stiffeningMean Young's Mean ultimate stress Modulus Intensity Increase % IntensityIncrease % Sample (MPa) (P value) (MPa) (P Value) In Control: untreated3.12 ± 0.73 12.69 ± 3.31 vivo fellow eyes n = 10 WST11 10 min treated4.51 ± 1.85 45 (0.1446) 13.9 ± 3.9 9 (0.183) eyes n = 4 WST11 20 mintreated 6.66 ± 0.67 113 (<0.0001)  22.7 ± 3.82 79 (0.0028  eyes n = 6

Notably, the in-vivo rabbits were 12 weeks old at the time of treatment,and 16 weeks old when sacrificed, while the ex-vivo group rabbits weresacrificed at the age of 12 weeks. The aging resulted in a higherbaseline Young's modulus and ultimate stress values of the control eyes(Table 1) that probably accounts for the gap between the parametersachieved in the two settings (Knox et al., 2010; Elsheikh et al., 2007).

Stress-strain measurements of rabbit cornea were conducted, both ex vivoand in vivo as described above, but exposing the eyes to salinesolutions of Compound 1-3 for the indicated times, followed by NIRillumination. The ultimate stress and Young's modulus values obtainedfor these compounds were similar to those obtained with WST11-S (datanot shown).

Example 4 In Vivo Cornea Treatment by WST11-D/NIR

To determine the role of dextran, the photochemical treatment with aformulation of WST11 2.5 mg/ml containing 20% dextran T-500 (WST11-D)was examined. Six rabbits were examined: 3 rabbits were pretreated for20 min with WST11-S solution and 3 with WST11-D solution, followed byNIR illumination for 30 minutes. Additional 4 rabbits were treated withRF (n=2) or RF-D (n=2) for 30 minutes, followed by 30-min UVAirradiation (370 nm, 3 mW/cm²).

As shown in FIGS. 10A-10B, application of WST11-S or WST11-D did notappear to affect the approximately two-fold increase in both the meanultimate stress and Young's modulus, as compared with untreated corneasof fellow eyes.

However, the important finding is that treatment with WST11significantly reduced the extent and duration of the edema andepithelial defect compared to RF-D/UVA treatment. Corneal edema clearedafter 5 days, and the epithelium healed within 7-9 days without hazedevelopment. In the RF and RF-D treated corneas, the epithelial defecthealed after 4 days. In the RF-D group the edema resolved after 4 dayswith recovery of transparency, whereas in the RF group the edemapersisted for 6 days, followed by 2 days of central epithelial haze.

Example 5 Endothelial and Keratocyte Response to WST11-S/NIR orWST11-D/NIR Treatment

The endothelial and keratocyte response of rabbits' eyes to incubationwith saline or dextran solutions of WST11 and Compounds 1-7, followed byNIR illumination were studied. The effects of these variousbactriochlorophyll solutions were practically the same. Detailed resultsare provided herein for measurements with WST11.

One rabbit underwent treatment with WST11 in saline (WST11-S; 20-minuteincubation, 30-minute irradiation at 755 nm). Six rabbits underwenttreatment with WST11D (n=4, 20-minute incubation, 30-minute irradiationat 755 nm), or with RF-D (n=2, 30-minute incubation, 30-minuteirradiation at 370 nm) as described above (In-vivo studies section).Contralateral eyes were used as control.

Histological examination of the corneas two days after WST11-S/NIRtreatment showed marked edema (cornea swelling to 890 μm), and a reducednumber of keratocytes throughout the stroma, more pronounced in theanterior half (see FIG. 1B). A honeycomb-like lacunar hydration pattern,containing keratocytes or keratocyte debris, was present. However, theendothelial cell layer appeared intact and did not differ from thecontrol (FIG. 11A). There was no statistical difference in theendothelial counts between treatment and control (P=0.47). In contrast,corneas treated with WST11-D showed minimal corneal edema, absence ofthe epithelium in the central area and a statistically significantreduction in the number of keratocytes (481±121 cells/mm² in the treatedcorneas, as compared to 1060±210 cells/mm² in controls, P<0.0001)limited to the outer half of the stroma (FIG. 11C). There was noevidence of damage to the endothelium in comparison to the control, bothafter vital (data not shown) and H&E staining (FIG. 11A). One week aftertreatment, the histological sections showed shrinkage of the anteriorstroma to 250 μm (in addition to the stromal compaction to 340 μm thatoccurred following formaldehyde fixation of control samples), loss ofkeratocytes in the anterior ⅓ of the stroma (80 μm) with epithelialhealing, but no endothelial damage (FIG. 11D). H&E staining of theretina two days after WST11-D/NIR treatment did not show anymorphological changes compared with control (data not shown).

Apoptosis was examined using peroxidase-based terminaldeoxyribonucleotidyl transferase-mediated dUTP-digoxigenin nick andlabeling (TUNEL) assay. One day postoperatively, TUNEL-positivekeratocytes were detected in the outer ½ of anterior stroma of treatedcorneas, as shown in FIG. 12B. No staining for TUNEL was observed in theposterior stroma the endothelium was absent and there was edema of thecentral stoma, as in the control corneas (FIG. 12A).

Example 6 Fluorescence Spectroscopy of Rabbit Corneas

Eight rabbit eyes were enucleated post mortem and corneal endotheliumwas de-epithlialized. Three eyes were immersed in SWT11-S solution and 3eyes in SWT11-D solution for 30 minutes, followed by NIR illumination.Two eyes were treated with RF-D solution for 30 minutes, followed by UVAirradiation. Contralateral eyes served as control. Fluorescence ofsegments of the corneas was measured as described in Materials andMethods.

Excitation of the RF-D/UVA treated corneas at 320 nm generated a clearemission signal at 405 nm that probably corresponded to the fluorescenceof dityrosine, a known signature of cross linking (Kato et al., 1994).Such emission was absent in the WST11-S/NIR and WST11-D/NIR treatedcorneas, and in the controls as shown in FIG. 13. Excitation at anyother wavelength did not provide any significant change in the corneaemission profile.

Example 7 Palladium Based Measurement of Systemic Absorption ofTopically Applied WST11-D

Six rabbits were anesthetized as described in the Materials and Methods.After de-epithelialization, one cornea of each rabbit was treated withWST11-D 2.5 mg/ml (n=3) and 10 mg/ml (n=3) for 20 minutes using an eyecap. Blood samples (˜0.5 ml) were taken from the ear vein beforeapplication (time 0), and at 10, 20, 40 and 60 minutes after applicationbegan, placed in pre-weighed polyethylene 1.5 ml test tubes, weightedand lyophilized. The dry samples were digested with nitric acid, and Pdconcentrations were determined by inductively-coupled plasma massspectrometry (ICP-MS) using a set of Pd standards (High-purityStandards, USA) as previously described (Mazor et al, 2005).

ICP-MS measurements of blood samples drawn from rabbits during and aftertopical application of WST11-D could not detect any significant levelsof Pd⁺², as an evidence for no penetration of the drug into thecirculation of the treated animals at all measured time points.

Example 8 Thermographic Analysis of the Corneal Surface

Three rabbits were treated with WST11-D for 20 minutes, followed by NIRillumination (755 nm, 10 mW/cm²) for 30 minutes. Temperaturemeasurements on the corneal surface were performed during WST11-D/NIRtreatment using an IR thermocamera (Thermal imager InfRec R300, NEC AvioInfrared Technologies Co., Ltd., Tokyo, Japan) with thermal resolution0.05° C., temperature accuracy of ±1° C. and a spatial resolution of 120μm. Images were recorded before irradiation, every 7 minutes duringirradiation and at the conclusion (last seconds) of irradiation.Selected thermographic images were processed with InfReC Analyzer NS9500Lite (NEC Avio Infrared Technologies Co., Ltd., Tokyo, Japan).

A constant temperature gradient from T=32° C. at the corneal center toT=37.5° C. at the limbal periphery was measured before, during and aftertreatment. Deviations of less than 1° C. were observed throughout thewhole procedure (data not shown).

Example 9 Transepithelial Delivery of WST11 into Rabbit Cornea

Permeability of the cornea to drugs is clinically important because itis the major factor determining the efficacy of topically appliedophthalmic formulations. To study the transepithelial penetration of theophthalmic formulations of the invention, the delivery of SWT11 wasassessed in the presence of a known transepithelial permeabilityenhancing excipient comprising benzalkonium chloride solution containingNaCl.

Two rabbits were anesthetized and treated as follows:

In one eye of each rabbit, the epithelium was removed, and the centralcorneal area was incubated with a 2.5 mg/ml solution of WST11 in 0.9%sodium chloride, pH 7.3, using eye cap during 20 minutes. In thecontralateral eye the epithelium was left intact and the central cornealarea was incubated with a 2.5 mg/ml solution of WST11 in 0.44% sodiumchloride containing 0.02% benzalkonium chloride, pH 7.3, using eye cap,during 20 minutes, and then the epithelium was removed.

-   -   Following incubation with the photosensitizer, the animals were        euthanized, central corneal discs of 8 mm in diameter were        removed, and photosensitizer's overall accumulation was        estimated by recording absorption spectra and measurement of OD        at 755 nm as described in Materials and Methods.

Accumulation of WST11 in the de-epithelialized corneas resulted inoptical density (OD) of about 0.86 in one rabbit and about 0.75 in thesecond rabbit. Transepithelial corneal accumulation resulted in OD ofabout 0.12 in the first rabbit which is about 14% of WST11 accumulatedin the de-epithelialized cornea, and about 0.16 in the second rabbit,which is about 21% of the photosensitizer that accumulated in thede-epithelialized cornea.

Thus, under the condition applied above, the amount of photosensitizerdelivered by transepithelial corneal incubation can be ˜⅙ of the amountaccumulated in de-epithelialized cornea.

Example 10 Penetration Depth in Cornea of WST11 in Aqueous 65% SucroseSolution

Aqueous 65% sucrose solution has similar viscosity as 20% dextran T-500(˜175 cP). The penetration depth of topically applied photosensitizer ina formulation comprising 65% sucrose was tested in corneas of rabbits.The results described herein were obtained for WST11 in 65% sucrosesolution.

Two rabbits were anesthetized, their corneas were de-epithelialized andone eye of each rabbit was treated with a 2.5 mg/ml solution of WST11 inaqueous 65% sucrose solution, pH 7.3, using eye cap, during 10 minutes,and the contralateral eye was similarly treated but for 30 minutes.Then, the animals were euthanized, central corneal discs of 8 mm indiameter were removed, deep frozen and cut for sagittal slices asdescribed in Materials and Methods.

After 10-min. incubation the photosensitizer penetrated to ˜⅓ of theouter stroma, and after 30-min. incubation to ˜½ of the outer stroma, inboth animals (data not shown). Thus, a 65% sucrose solution can beapplied as effectively as dextran for limiting photosensitizerpenetration into cornea.

Example 11 WST11 Depth of Penetration into the Rabbit Sclera

The depth of penetration of WST11 into rabbit sclera was assessed byfluorescence microscopy as described in Materials and Methods. Theresults shown in FIG. 14 indicate that WST11 applied ex vivo using eyecap and in-situ using Merocel® sponge penetrated into sclera tissue.

Example 12 Biomechanical Testing of Rabbit Sclera Treated Ex Vivo withWST11-S and External NIR Illumination

Stress-strain tests of sclera of enucleated rabbits' eyes were performedfollowing treatment with WST11 (2.5 mg/ml) as described in Materials andMethods. After photosensitizer impregnation, the sclera was illuminatedby applying the laser beam directly onto the treated area (externalillumination), using a flat optical fiber that run along a curvedplastic or metal glide as shown in FIG. 17A. Strips of the sclera werecut and tested.

The measurements demonstrated a significant increase in the stiffness ofthe treated sclera. The mean maximal stress in the control sclera was2.77±0.99 MPa. The mean maximal stress in WST11/NIR treated sclera was7.6±0.99 MPa (174% increase). The mean Young's modulus in control sclerawas 15.2±7.5 MPa. The mean Young's modulus in WST11/NIR treated sclerawas 45.6±3.9 MPa (200% increase). The results are shown in FIGS.15A-15B.

Example 13 Biomechanical Testing of Rabbit Sclera Treated Ex Vivo withWST11-S and NIR Illumination Through the Anterior Eye Segment

The inventors hypothesized that since tissues are fairly transparent toillumination at 755-820 nm, it should be possible to deliver sufficientillumination to the posterior sclera by NIR illumination through thecornea and anterior segment of the eye using either the three mirrorfundus lens apparatus shown in FIGS. 18A-18C, or by illuminating thecornea through an indirect ophthalmoscope using the apparatus describedin FIG. 17B.

Six eyes of three rabbits were enucleated. The posterior eye area up tothe equatorial line was incubated using an eye cap with 2.5 mg/mlsolution of WST11 in saline pH 7.3 for 20 minutes and then illuminatedby NIR illumination through the anterior cornea using a diode laser (755nm, 0.5 W) and three mirror fundus lens (FIGS. 18A-18C) for 30 minutes.Strips of sclera were cut and tested as described in Materials andMethods.

The measurements demonstrated an increase in the stiffness of thetreated sclera. The mean maximal stress in control sclera was 4.65±1.15MPa. The mean maximal stress in WST11/NIR treated corneas was 6.59±1.32MPa (42% increase). The mean Young's modulus in control sclera was25.25±5.30 MPa. The mean Young's modulus in WST11/NIR treated sclera was38.83±6.89 MPa (54% increase). The results are shown in FIGS. 16A-16B

Example 14 In Vivo Sclera Stiffening with Illumination Through theAnterior Segment

In vivo scleral stiffening while avoiding retinal and eye orbit toxicityis achieved as follows:

Impregnation of the photosensitizer (a Bchl derivative) into the sclerais performed by inserting a sub-tenon glide through openings in thelimbal conjunctiva in 4 quadrants. The inner distal 10 mm of the glideis porous and contains a reservoir of the test compound. This reservoiris connected by a tube that runs outside along the glide. After placingthe glide attached to the sclera, the tube is connected to a syringe orpump, and the test compound reservoir is injected to impregnate thesclera. The sensitizer concentration and time of exposure is a priorioptimized via ex-vivo measurements of enucleated rabbit eyes usingfluorescence spectroscopy of frozen histological sclera sections asdescribed in Example 11 above. If required, dextran or sucrose solutionsare used to optimize penetration depth.

The retina safety depends on the traversing light energy. In preclinicaland Phase II clinical trials of vascular targeted PDT with WST11, theinventors have shown that illumination of retina by 50-70 J (82-120 secat 600 mW/cm²) proved safe. Consequently, the treatment energy is set atthe range of 5-12 J (10-20 mW/cm² for 20-10 min) for which minimalmorbidity of the retina is expected.

Using the optimized parameters Bchl-S or Bchl-D is applied as describedabove followed by 755-nm frontal illumination at an optimized laserpower output in the range of 20-250 mW to deliver an a priori optimizedlight intensity at the posterior segment of the eye. The illumination isperformed using a NIR laser with He—Ne aiming beam through Goldmann 3mirror fundus lens or NIR laser attached to an indirect ophthalmoscopewith He—Ne aiming beam. One month after the treatment the rabbits areeuthanized and the eyes are enucleated. Treatment success is assessed bystrain-stress measurements of scleral strips excised from the superiorand the inferior sclera of the treated eyes compared to the matchingsclera of non-treated fellow eyes.

Example 15 In Vivo Sclera Stiffening with Illumination Through theAnterior Segment

In vivo scleral stiffening while avoiding retinal and eye orbit toxicityis achieved as described in Example 14, but the photosensitizerdelivered to the sclera via a Merocel® sponge attached to the glideinserted through openings in the limbal conjunctiva, instead of using aporous glide to deliver the photosensitizer.

Example 16 In Vivo Sclera Stiffening with Illumination Through theAnterior Segment

In vivo scleral stiffening is achieved as described in Example 14, butillumination is applied externally by insertion of a glide connected tooptical fiber through the limbal conjunctiva openings as illustrated in(FIG. 17B). This glide is designed to spread a diffuse light directedtowards the sclera; it is opaque in its orbital aspect. The illuminationglide is connected to a NIR laser or LED source to deliver the NIRlight. Both impregnation and illumination glides are marked with amillimetric ruler to verify insertion depth. Position of illuminationglide can be monitored during light delivery by fundoscopy or fundusimaging.

An alternative glide that is used for in vivo scleral stiffening is aglide designed as a combined unit for both drug and illuminationdelivery.

REFERENCES

-   Ashur I, et al. 2009, “Photocatalytic generation of oxygen radicals    by the water-soluble bacteriochlorophyll derivative WST11,    noncovalently bound to serum albumin”, J Phys Chem., 113:8027-37.-   Avila M Y, Navia J L, 2010, “Effect of genipin collagen crosslinking    on porcine corneas”, J Cataract Refract Surg., 36:659-664.-   Berdugo M et al. 2008, “Evaluation of the new photosensitizer stakel    (WST-11) for photodynamic choroidal vessel occlusion in rabbit and    rat eyes”, Inv Ophthalmol Vis Sci. 49:1633-1644.-   Bourges J L et al., 2006, “PDT of corneal neovessels using a new    hydrosoluble photosensitizer (WST11)”, Acta Ophthalmol Scand., 84(S    239:41): 352.-   Brandis A, Mazor O, Neumark E, Rosenbach-Belkin V, Salomon Y,    Scherz A. 2005, “Novel water-soluble bacteriochlorophyll derivatives    for vascular-targeted photodynamic therapy: synthesis, solubility,    phototoxicity, and the effect of serum proteins”, Photochem    Photobiol., 81:983-993.-   Elsheikh A, Wang D, Brown M, Rama P, Campanelli M, Pye D, 2007,    “Assessment of corneal biomechanical properties and their variation    with age”, Curr Eye Res., 32:11-19.-   Hafezi F, Kanellopoulos J, Wiltfang R, Seiler T. 2007, “Corneal    collagen crosslinking with riboflavin and ultraviolet A to treat    induced keratoectasia after laser in situ keratomileusis” J Cataract    Refract Surg., 33:2035-2040.-   Hafezi F, Mrochen M, Iseli H P, Seiler T. 2009, “Collagen    crosslinking with ultraviolet-A and hypoosmolar riboflavin solution    in thin corneas”, J Cataract Refract Surg., 35:621-624.-   Kato Y, Uchida K, Kawakishi S. Aggregation of collagen exposed to    UVA in the presence of riboflavin: a plausible role of tyrosine    modification. Photochem Photobiol. 1994; 59:343-349.-   Knox Cartwright N E, Tyrer J R, Marshall J, 2010, “Age-related    differences in the elasticity of the human cornea”, Invest    Ophthalmol Vis Sci., 52:4324-4329.-   Lepor H. 2008, “Vascular targeted photodynamic therapy for localized    prostate cancer”, Rev Urol., 10:254-261.-   Letko E, Majmudar P A, Forstot S L, Epstein R J, Rubinfeld R S,    2011, “UVA-light and riboflavin-mediated corneal collagen    cross-linking”, Int ophthalmol clin., 51:63-76.-   Liu K et al. 2004, “Superoxide, hydrogen peroxide and hydroxyl    radical in D1/D2/cytochrome b-559 Photosystem II reaction center    complex”, Photosynthesis Research., 81:41-47.-   Mazor O. et al. 2005, “WST11, A novel water-soluble    bacteriochlorophyll derivative; cellular uptake, pharmacokinetics,    biodistribution, and vascular targeted photodynamic activity against    melanoma tumors”, Photochem Photobiol., 81:342-345.-   Moore C M, Pendse D, Emberton M. 2009, “Photodynamic therapy for    prostate cancer-a review of current status and future promise”, Nat    Clin Pract Urol., 6:18-30.-   Raiskup-Wolf F, Hoyer A, Spoerl E, Pillunat L E. 2008, “Collagen    cross-linking with riboflavin and ultraviolet-A light in    keratoconus: long-term results”, J Cataract Refract Surg. 34:796-801-   Søndergaard A P, Hjortdal J, Breitenbach T, Ivarsen A, 2010,    “Corneal distribution of riboflavin prior to collagen    cross-linking”, Curr Eye Res., 116:121-135.-   Spence D J, Peyman G A. 1976, “A new technique for the vital    staining of the corneal endothelium”, Inv Ophthalmol Vis Sci., 15:    1000-1002.-   Spoerl E, Mrochen M, Sliney D, Trokel S, Seiler T. 2007, “Safety of    UVA-riboflavin cross-linking of the cornea”, Cornea, 26:385-389.-   Trachtenberg J et al. 2007, “Vascular targeted photodynamic therapy    with palladium-bacteriopheophorbide photosensitizer for recurrent    prostate cancer following definitive radiation therapy: assessment    of safety and treatment response”, J Urol., 178:1974-1979.-   Vakrat-Haglili Y et al., 2005, “The microenvironment effect on the    generation of reactive oxygen species by Pd-Bacteriopheophorbide”, J    Am Chem Soc., 127:6487-6497.-   Wollensak G, 2010(a) “Histological changes in human cornea after    cross-linking with riboflavin and ultraviolet A”, Letter to the    editor. Acta Ophthalmol. 88:e17-18.-   Wollensak G, Aurich H, Wirbelauer C, Saadettin S. 2010(b),    “Significance of the riboflavin film in corneal collagen    crosslinking”, J Cataract Refract Surg., 36:114-120.-   Wollensak G, Spoerl E, Reber F, Pillunat L, Funk R. 2003(c),    “Corneal endothelial cytoxicity of riboflavin/UVA treatment in    vitro”, Ophthalmic Res., 35:324-328.-   Wollensak G, Spoerl E, Reber F, Seiler T. 2004(a) “Keratocyte    cytotoxicity of riboflavin/UVA-treatment in vitro”, Eye, 18:718-722.-   Wollensak G, Spoerl E, Seiler T. 2003(a)    “Riboflavin/ultraviolet-A-induced collagen crosslinking for the    treatment of keratoconus”, Am J Ophthalmol, 135:620-627.-   Wollensak G, Spoerl E, Wilsch M, Seiler T. 2003(b) “Endothelial cell    damage after riboflavin-ultraviolet-A treatment in the rabbit”, J    Cataract Refract Surg. 29:1786-1790.-   Wollensak G, Spoerl E, Wilsch M, Seiler T. 2004(b) “Keratocyte    apoptosis after corneal collagen cross-linking using riboflavin/UVA    treatment”, Cornea. 23:43-49.

The invention claimed is:
 1. A method of treating keratoconus byphotodynamic therapy (PDT), said method comprising the steps of: (i)administering to an eye of an individual in need thereof an effectiveamount of Palladium 3¹-oxo-15-methoxycarbonylmethyl-rhodobacteriochlorin13¹-(2-sulfoethyl)amide, or a pharmaceutically acceptable salt thereof,and (ii) irradiating the eye of said individual with light at a red ornear infrared (NIR) wavelength.
 2. The method according to claim 1,wherein the pharmaceutically acceptable salt thereof is a potassium saltof Palladium 3¹-oxo-15-methoxycarbonylmethyl-rhodobacteriochlorin13¹-(2-sulfoethyl)amide.
 3. The method according to claim 2, wherein thepotassium salt of Palladium3¹-oxo-15-methoxycarbonylmethyl-rhodobacteriochlorin13¹-(2-sulfoethyl)amide is Palladium3¹-oxo-15-methoxycarbonylmethyl-rhodobacteriochlorin13¹-(2-sulfoethyl)amide dipotassium salt.
 4. The method according toclaim 1, wherein the pharmaceutically acceptable salt thereof is a saltof Palladium 3¹-oxo-15-methoxycarbonylmethyl-rhodobacteriochlorin13¹-(2-sulfoethyl) amide with a monovalent alkaline metal or ammonium.5. The method according to claim 4, wherein the pharmaceuticallyacceptable salt thereof is a salt of Palladium3¹-oxo-15-methoxycarbonylmethyl-rhodobacteriochlorin 13¹-(2-sulfoethyl)amide with a monovalent alkaline metal selected from the groupconsisting of potassium, sodium and lithium.